Tumor necrosis factor receptor-associated factors

ABSTRACT

The invention concerns new tumor necrosis factor receptor associated factors, designated TRAF. The new factors are capable of specific association with the intracellular domain of the type 2 TNF receptor (TNF-R2), and are involved in the mediation of TNF biological activities.

FIELD OF THE INVENTION

[0001] The present invention concerns novel polypeptide factors. Moreparticularly, the invention concerns factors associated with the type 2tumor necrosis factor receptor (TNF-R2).

BACKGROUND OF THE INVENTION

[0002] Tumor necrosis factor (TNF, also referred to as TNF-α) is apotent cytokine produced mainly by activated macrophages and a few othercell types. The large number of biological effects elicited by TNFinclude hemorrhagic necrosis of transplanted tumors, cytotoxicity, arole in endotoxin shock, inflammatory, immunoregulatory, proliferative,and antiviral responses [reviewed in Goeddel, D. V. et al., Cold SpringHarbor Symposia on Quantitative Biology 51, 597-609 (1986); Beutler, B.and Cerami, A., Ann. Rev. Biochem. 57, 505-518 (1988); Old, L. J., Sci.Am. 258(5), 59-75 (1988); Fiers, W. FEBS Lett. 285(2), 199-212 (1991)].The literature has reported that TNF and other cytokines such as IL-1may protect against the deleterious effects of ionizing radiationproduced during the course of radiotherapy, such as denaturation ofenzymes, lipid peroxidation, and DNA damage [(Neta et al., J. Immunol.136(7): 2483, (1987); Neta et al., Fed. Proc. 46: 1200 (abstract),(1987); Urbaschek et al., Lymphokine Res.6: 179 (1987); U.S. Pat. No.4,861,587; Neta et al., J. Immunol. 140: 108 (1988)]. A relatedmolecule, lymphotoxin (LT, also referred to as TNF-β), that is producedby activated lymphocytes shows a similar but not identical spectrum ofbiological activities as TNF (see, e.g. Goeddel, D. V. et al., supra,and Fiers, W., supra). TNF was described by Pennica et al., Nature 31 2,721 (1984); LT was described by Gray et al., Nature 312, 724 (1984).

[0003] The first step in the induction of the various cellular responsesmediated by TNF or LT is their binding to specific cell surfacereceptors. Two distinct TNF receptors of approximately 55-kDa (TNF-R1)and 75-kDa (TNF-R2) have been identified [Hohmann, H. P. et al., J.Biol. Chem. 264 14927-14934 (1989); Brockhaus, M. et al., Proc. Natl.Acad. Sci. USA 87, 3127-3131 (1990)], and human and mouse cDNAscorresponding to both receptor types have been isolated andcharacterized [Loetscher, H. et al., Cell 61, 351 (1990); Schall, T. J.et al., Cell 61, 361 (1990); Smith, C. A. et al., Science 248, 1019(1990); Lewis, M. et al., Proc. Natl. Acad. Sci. USA 88, 2830-2834(1991); Goodwin, R. G. et al., Mol. Cell. Biol. 11, 3020-3026 (1991)].Both TNF-Rs share the typical structure of cell surface receptorsincluding extracellular, transmembrane and intracellular regions. Theextracellular portions of both receptors are found naturally also assoluble TNF-binding proteins [Nophar, Y. et al., EMBO J. 9, 3269 (1990);and Kohno, T. et al., Proc. Natl. Acad. Sci. U.S.A. 87, 8331 (1990)]].The amino acid sequence of human TNF-R1 and the underlying nucleotidesequence are disclosed in EP 417,563 (published Mar. 20, 1991), whereasEP 418,014 (published Mar. 20, 1991) discloses the amino acid andnucleotide sequences of human TNF-R2.

[0004] Although not yet systematically investigated, the majority ofcell types and tissues appear to express both TNF receptors.

[0005] The individual roles of the two TNF receptors, and particularlythose of TNF-R2, in cell signaling are far from entirely understood,although studies performed by poly- and monoclonal antibodies (mAbs)that are specific for either TNF-R1 or TNF-R2 have provided some veryvaluable insight into the functions and interactions of these receptors.

[0006] It has been observed that both polyclonal and monoclonalantibodies directed against TNF-R1 can act as specific agonists for thisreceptor and elicit several TNF activities such as cytotoxicity,fibroblast proliferation, resistance to chlamydiae, and synthesis ofprostaglandin E₂ [Engelmann, H. et al., J. Biol. Chem. 265, 14497-14504(1990); Espevik, T. et al., J. Exp. Med. 171, 415-426 (1990); Shalaby,M. R. et al., J. Exp. Med. 172, 1517-1520 (1990)]. Agonist antibodies toTNF-R1 with antiviral activity are disclosed in copending applicationSer. No.07/856,989 filed Mar. 24, 1992.

[0007] In addition, polyclonal antibodies to both murine TNF-R1 andTNF-R2 have been developed, have been shown to behave as specificreceptor agonists and induce a subset of murine TNF activities. Whilethe murine TNF-R1 was shown to be responsible for signaling cytotoxicityand the induction of several genes, the murine TNF-R2 was shown to becapable of signaling proliferation of primary thymocytes and a cytotoxicT cell line, CT6 [Tartaglia, L. A. et al., Proc. Natl. Acad. Sci. USA88, 9292-9296 (1991)]. The ability of TNF-R2 to stimulate humanthymocyte proliferation has been demonstrated in experiments withmonoclonal antibodies directed against the human receptor.

[0008] Monoclonal antibodies against human TNF-R1 that block the bindingof TNF to TNF-R1 and antagonize several of the TNF effects have alsobeen described [Espevik, T. et al., Supra; Shalaby, M. R. et al., Supra;Naume, B. et al., J. Immunol. 146, 3035-3048 (1991)].

[0009] In addition, several reports described monoclonal antibodiesdirected against TNF-R2 that can partially antagonize the same TNFresponses (such as cytotoxicity and activation of NF-κB) that areinduced by TNF-R1 agonists [Shalaby, M. R. et al., Supra; Naume, B. etal., Supra; and Hohmann, H. P. et al., J. Biol. Chem. 265, 22409-22417(1990)].

[0010] It is now well established that although the two human TNFreceptors are both active in signal transduction, they are able tomediate distinct cellular responses. While TNF-R1 appears to beresponsible for signaling most TNF responses, the thymocyteproliferation stimulating activity of TNF is specifically mediated byTNF-R2. In addition, TNF-R2 activates the transcription factor NF-κB(Lenardo & Baltimore, Cell 58: 227-229 [1989]) and mediates thetranscriptional induction of the granulocyte-macrophage colonystimulating factor (GM-CSF) gene (Miyatake et al., EMBO J. 4: 2561-2568[1985]; Stanley et al., EMBO J. 4: 2569-2573 [1985]) and the A20 zincfinger protein gene (Opipari et al., J. Biol. Chem. 265: 14705-14708[1990]) in CT6 cells. TNF-R2 also participates as an accessory componentto TNF-R1 in the signaling of responses primarily mediated by TNF-R1,like cytotoxicity ([Tartaglia, L. A. and Goeddel, D. V., Immunol. Today13, 151-153 [1992]).

SUMMARY OF THE INVENTION

[0011] Although TNF itself, the TNF receptors and TNF activitiesmediated by the two receptors have been studied extensively, thepost-receptor signal transduction mechanisms are unknown (see the reviewarticle by Beyaert, R. & Fiers, W., “Molecular mechanisms of tumornecrosis factor-induced cytotoxicity: what we do understand and what wedo not”, FEBS Letters 340, 9-16 (1994)). This is especially true for thevery first step in the TNF receptor signal transduction cascade, i.e.for the question of how the membrane-bound receptor sends a signal intothe cell after activation by the ligand, TNF.

[0012] The present invention is based on the hypothesis that polypeptidefactors associated with the intracellular domain of TNF-R2 exist andparticipate in the TNF-R2 signal transduction cascade. Morespecifically, this invention is based on research directed to theidentification and isolation of native polypeptide factors that arecapable of association with the intracellular domain of TNF-R2 andparticipate in the intracellular post-receptor signaling of TNFbiological activities.

[0013] It is known that the TNF induced proliferation of murine CT6cells is mediated by TNF-R2 (Tartaglia et al., [1991], supra). Toidentify factors that are associated with the intracellular domain ofhTNF-R2, the receptor was immunoprecipitated from lysates of[³⁵S]-labeled transfected CT6 cells and from unlabeled transfected humanembryonic kidney 293 cells, which were then incubated with labeledlysate from untransfected CT6 cells. Several polypeptides with apparentmolecular weights of 45-50 kD and one with an approximate molecularweight of 68 kD were specifically coprecipitated with theimmunoprecipitated hTNF-R2. These are hereinafter collectively referredto as tumor necrosis factor receptor associated polypeptides, or TRAFs.Of the factors identified two have so far been purified and cloned.These two factors are designated as tumor necrosis factor receptorassociated factors 1 and 2 (TRAF1 and TRAF2; SEQ. ID. NOs: 2 and 4). Acomparison of the amino acid sequences of TRAF1 and TRAF2 revealed thatthey share a high degree of amino acid identity in their C-terminaldomains (53% identity over 230 amino acids), while their N-terminaldomains are unrelated. These new factors are believed to play a key rolein the post-receptor signaling of TNF. Since the intracellular domain ofTNF-R2 does not display any sequence homology to any other knownreceptor or protein, these signaling molecules might represent a novelsignal transduction mechanism, the understanding of which can greatlycontribute to the development of new strategies to improve thetherapeutic value of TNF.

[0014] In one aspect, the present invention concerns a family of novelfactors (TRAFs) capable of specific association with the intracellulardomain of a native TNF-R2. The invention specifically concerns tumornecrosis factor receptor associated factors 1 and 2 (TRAF1 and TRAF2,SEQ. ID. NOs. 2 and 4), including the native factors from any human ornon-human animal species and their functional derivatives.

[0015] In another aspect, the invention concerns an isolated nucleicacid molecule comprising a nucleotide sequence encoding a TRAFpolypeptide.

[0016] In yet another aspect, the invention concerns an expressionvector comprising the foregoing nucleic acid molecule operably linked tocontrol sequences recognized by a host cell transformed with the vector.

[0017] In a further aspect, the invention concerns a host celltransformed with the foregoing expression vector.

[0018] In a still further aspect, the invention concerns molecules(including polypeptides, e.g. antibodies and TRAF analogs and fragments,peptides and small organic molecules) which disrupt the interaction of aTNF-R2 receptor associated factor and TNF-R2.

[0019] The invention specifically concerns antibodies, capable ofspecific binding to a native TRAF polypeptide, and hybridoma cell linesproducing such antibodies.

[0020] In a different aspect, the invention concerns a method of using anucleic acid molecule encoding a TRAF polypeptide as hereinabovedefined, comprising expressing such nucleic acid molecule in a culturedhost cell transformed with a vector comprising said nucleic acidmolecule operably linked to control sequences recognized by the hostcell transformed with the vector, and recovering the encoded polypeptidefrom the host cell.

[0021] The invention further concerns a method for producing a TRAFpolypeptide as hereinabove defined, comprising inserting into the DNA ofa cell containing nucleic acid encoding said polypeptide a transcriptionmodulatory element in sufficient proximity and orientation to thenucleic acid molecule to influence the transcription thereof.

[0022] The invention also provides a method of determining the presenceof a TRAF polypeptide, comprising hybridizing DNA encoding suchpolypeptide to a test sample nucleic acid and determining the presenceof TRAF polypeptide DNA.

[0023] In a further aspect, the invention concerns an isolated nucleicacid molecule encoding a fusion of an intracellular domain sequence of anative TNF-R2 and the DNA-binding domain of a transcriptional activator.

[0024] In a still further aspect, the invention concerns an isolatednucleic acid molecule encoding a fusion of a TRAF to the activationdomain of a transcriptional activator.

[0025] The invention further concerns hybrid (fusion) polypeptidesencoded by the foregoing nucleic acids.

[0026] The invention also covers vectors comprising one or both of thenucleic acid molecules encoding the foregoing fusion proteins.

[0027] In a different aspect, the invention concerns an assay foridentifying a factor capable of specific binding to the intracellulardomain of a native TNF-R2, comprising

[0028] (a) expressing, in a single host cell carrying a reporter gene,nucleic acid molecules encoding a polypeptide comprising a fusion of anintracellular domain sequence of a native TNF-R2 to the DNA-bindingdomain of a transcriptional activator, and a fusion of a candidatefactor to the activation domain of a transcriptional activator; and

[0029] (b) monitoring the binding of the candidate factor to the TNF-R2intracellular domain sequence by detecting the molecule encoded by thereporter gene.

[0030] The invention further relates to an assay for identifying afactor capable of specific association with the intracellular domain ofa native TNF-R2, comprising

[0031] (a) expressing nucleic acid molecules encoding a polypeptidecomprising a fusion of an intracellular domain sequence of a nativeTNF-R2 to the DNA-binding domain of a transcriptional activator, and asecond polypeptide comprising a fusion of a candidate polypeptide factorto the activation domain of a transcriptional activator, in a singlehost cell transfected with nucleic acid encoding a polypeptide factorcapable of specific binding to said TNF-R2, and with nucleic acidencoding a reporter gene; and

[0032] (b) monitoring the association of said candidate factor with saidTNF-R2 or with said polypeptide factor capable of specific binding tosaid TNF-R2 by detecting the polypeptide encoded by said reporter gene.

[0033] In a further aspect, the invention concerns an assay foridentifying a molecule capable of disrupting the association of a TRAFwith the intracellular domain of a native TNF-R2, comprising contactinga cell expressing 1. a fusion of an intracellular domain sequence of anative TNF-R2 to the DNA-binding domain of a transcriptional activator,2. a fusion of a native TRAF polypeptide to the activation domain ofsaid transcriptional activator, and 3. a reporter gene, with a candidatemolecule, and monitoring the ability of said candidate molecule todisrupt the association of said TRAF and TNF-R2 intracellular domainsequence by detecting the molecule encoded by the reporter gene. Thecell, just in the previous assays is preferably a yeast cell.

[0034] In addition to the “two-hybrid” format described above, the assaymy be performed in any conventional binding/inhibitor assay format. Forexample, one binding partner (TNF-R2 or TRAF) may be immobilized, andcontacted with the other binding partner equipped with a detectablelabel, such as a radioactive label, e.g. ³²P and the binding(association) of the two partners is detected in the presence of acandidate inhibitor. The design of a specific binding assay is wellwithin the skill of a person skilled in the art.

[0035] In a different aspect, the invention concerns a method ofamplifying a nucleic acid test sample comprising priming a nucleic acidpolymerase reaction with nucleic acid encoding a TRAF polypeptide, asdefined above.

[0036] In another aspect, the invention concerns a method for detectinga nucleic acid sequence coding for a polypeptide molecule whichcomprises all or part of a TRAF polypeptide or a related nucleic acidsequence, comprising contacting the nucleic acid sequence with adetectable marker which binds specifically to at least part of thenucleic acid sequence, and detecting the marker so bound.

[0037] In yet another aspect, the invention concerns a method fortreating a pathological condition associated with a TNF biologicalactivity mediated, fully or partially, by TNF-R2, comprisingadministering to a patient in need a therapeutically effective amount ofa TRAF or a molecule capable of disrupting the interaction of a TRAF andTNF-R2.

BRIEF DESCRIPTION OF DRAWINGS

[0038]FIG. 1. Activation of the transcription factor NF-kB throughTNF-R2 in CT6 cells.

[0039] 6 μg of nuclear extract prepared from CT6 cells that had beenstimulated for 20 min with a 1:500 dilution of anti-mTNF-R2 polyclonalantibodies or the respective preimmune serum were incubated with aradiolabeled double-stranded oligonucleotide containing either twowild-type (wt) or mutant (mt) NF-kB binding sites and analyzed for theinduction of NF-kB DNA-binding activity by electrophoretic mobilityshift assay (Schütze et al., Cell 71, 765-776 [1992]). Binding reactionswere either performed without competitor oligonucleotides or in thepresence of a 500 fold excess of unlabeled competitor oligonucleotidescontaining mutant NF-kB binding sites or a binding site for thetranscription factor AP-1 (Angel, P. et al., Mol. Cell. Biol. 7:2256-2266 [1987]). F and B refer to free oligonucleotide probe andoligonucleotide probe in a complex with protein, respectively.

[0040]FIG. 2. Immunoprecipitation of hTNF-R2.

[0041] (A) ³⁵S-labeled CT6 cells or CT6 cells expressing the hTNF-R2were stimulated for 10 min with 100 ng/ml hTNF or left untreated. Thecells were lysed and the hTNF-R2 immunoprecipitated as described in thetext and analyzed by SDS-PAGE and autoradiography. The asterisk marksthe band corresponding to the 75-80 kd hTNF-R2.

[0042] (B) The hTNF-R2 was immunoprecipitated from unstimulated orTNF-stimulated 293 or 293/TNF-R2 cells and incubated with lysates from³⁵S-labeled CT6 cells. Arrows indicate bands of 45-50 kd and 68 kd thatcoprecipitate specifically with the hTNF-R2 in both experiments.Molecular weight markers are indicated on the right in kd.

[0043]FIG. 3. Purification of GST-hTNF-R2icd fusion protein.

[0044] Glutathione-S-transferase (GST) and GST-hTNF-R2icd fusion proteinwere expressed in E. coli, purified as described in the text andanalyzed by SDS-PAGE and Coomassie staining. Molecular weight markersare indicated on the right in kd.

[0045]FIG. 4. Coprecipitation of GST-hTNF-R2icd fusion protein in CT6cell extracts.

[0046] GST and GST-hTNF-R2icd fusion protein beads were incubated withlysates from ³⁵S-labeled CT6 cells as described in the text and analyzedby SDS-PAGE and autoradiography. Arrows indicate bands of 45-50 kd and68 kd that coprecipitate specifically with the GST-hTNF-R2icd fusionprotein. Molecular weight markers are indicated on the right in kd.

[0047]FIG. 5. Coprecipitation of GST-mutant hTNF-R2icd fusion proteinsin CT6 cell extracts.

[0048] GST and GST-fusion proteins containing mutant intracellulardomains of the hTNF-R2 were coupled to glutathione-agarose beads,incubated with lysates from ³⁵S-labeled CT6 cells and analyzed bySDS-PAGE and autoradiography. Arrows indicate bands of 45-50 kd and 68kd that coprecipitate specifically with the GST-fusion proteinscontaining the wild type (wt), the mutant −16, the Δ304-345 and the384-424 intracellular domains of hTNF-R2 but are not associated with themutant −37 and −59 intracellular domains. Note that the pattern of thesebands is compressed in some cases due to the unlabeled fusion proteinsmigrating at the same size. Molecular weight markers are indicated onthe right in kd.

[0049]FIG. 6. Competition of TNF-R2 associated factors withGST-hTNF-R2icd fusion proteins. p1 (A) The hTNF-R2 wasimmunoprecipitated from 293 and 293/TNF-R2 cells and incubated withlysates from ³⁵S-labeled CT6 cells that had been preincubated with 50 μlof the indicated GST-hTNF-R2icd fusion protein beads as competitor.Reactions were analyzed by SDS-PAGE and autoradiography. Arrows indicatebands of 45-50 kd and 68 kd that coprecipitate specifically with thehTNF-R2 and that are depleted by preincubation with GST-fusion proteinscontaining the wild type (wt) and the mutant −16 intracellular domainsof hTNF-R2 but not by preincubation with the mutant −37 and −59intracellular domain fusion proteins.

[0050] (B) The 68 kd region of a similar experiment as described in (A)is shown. Molecular weight markers are indicated on the right in kd.

[0051]FIG. 7. Coprecipitation of GST-hTNF-R2icd fusion protein in Jurkatcell extracts.

[0052] GST and GST-hTNF-R2icd fusion protein beads were incubated withlysates from ³⁵S-labeled Jurkat cells that had been stimulated for 10min with 100 ng/ml hTNF or left untreated. Reactions were analyzed bySDS-PAGE and autoradiography. Arrows indicate bands of 45-50 kd, 67 kdand 73-75 kd that coprecipitate specifically with the GST-hTNF-R2icdfusion protein. Molecular weight markers are indicated on the right inkd.

[0053]FIG. 8. Subcellular localization of TNF-R2 associated factors.

[0054] Cytoplasmic and cell membrane fractions were prepared from³⁵S-labeled CT6 cells as described in the text. These fractions and adetergent (total) extract form CT6 cells were incubated with GST andGST-hTNF-R2icd fusion beads, and the reactions analyzed by SDS-Page andautoradiography. Arrows indicate bands of 45-50 kd and 68 kd thatcoprecipitate specifically with the GST-hTNF-R2icd fusion protein.Molecular weight markers are indicated on the right in kd.

[0055]FIG. 9. Purification of TNF-R2 associated factors.

[0056] Large scale purification of TNF-R2 associated factors from CT6cells by GST-hTNF-R2icd fusion protein affinity chromatography wasperformed as described in the text. One tenth of the obtained materialwas analyzed by SDS-PAGE and silver staining. Arrows indicate bands of45-50 kd and 68-70 kd that were eluted specifically from theGST-hTNF-R2icd fusion protein affinity column. Molecular weight markersare indicated on the right in kd.

[0057]FIG. 10. Nucleotide and predicted amino acid sequence of the TRAF1cDNA (SEQ. ID. NOS: 1 and 2).

[0058] The nucleic acid sequence of the TRAF1 cDNA is shown withnumbering starting from the first base after the SalI cloning linker.The deduced protein sequence is displayed above with numbering from theinitiation methionine. In-frame stop codons upstream of the initiationmethionine are underlined. Amino acids identified by sequencing thepurified TRAF1 protein are indicated in bold. The TRAF domain (see text)comprises amino acids 180(>)-409(<). The potential leucine zipper region(see text) extends between amino acids 183(+)-259(−). Amino acids withinthis region defining the heptade motif are indicated in italic.

[0059]FIG. 11. Nucleotide and predicted amino acid sequence of the TRAF2cDNA (SEQ. ID. NOS: 3 and 4).

[0060] The nucleic acid sequence of the longest TRAF2 cDNA is shown withnumbering starting from the first base after the SalI cloning linker. Inaddition, the first nucleotide of four independently isolated pPC86 cDNAinserts (*) and the longest λ phage cDNA insert (^ ) is indicated. Thededuced protein sequence is displayed above with numbering from theputative initiation methionine, which is in-frame with the GAL4activation domain coding region in all isolated pPC86TRAF2 cDNA clones(see text). Cysteine and histidine residues defining the RING fingermotif and the two TFIIIA-like zinc finger motifs (see text) areindicated in bold or underlined, respectively. The TRAF domain (seetext) comprises amino acids 272(>)-501(<). The potential leucine zipperregion (see text) extends between amino acids 275(+)-351(−). Amino acidswithin this region defining the heptade motif are indicated in italic.

[0061]FIG. 12. Sequence similarity of regions in TRAF2 to zinc-bindingmotifs.

[0062] (A) Comparison of amino acid sequences containing RING fingermotifs. The TRAF2 RING finger motif is aligned with the respectivezinc-binding motifs of the regulatory protein COP1 from A. thaliana(Deng et al., Cell 71, 791-801 [1992]; SEQ. ID. NO: 5), the humanestrogen-responsive finger protein EFP (Inoue et al., Proc. Natl. Acad.Sci. USA 90, 11117-11121 [1993]; SEQ. ID. NO: 6), the RAD18 and UVS-2gene products required for DNA repair in S. cerevisiae and N. crassa,respectively (Jones et al., Nucl. Acids Res. 16, 7119-7131 [1988]; SEQ.ID. NO: 7; Tomita et al., Mol. Gen. Genet. 238, 225-233 [1993]; SEQ. ID.NO: 8), the human V(D)J recombination activating gene product RAG-1(Schatz et al., Cell 59, 1035-1048 [1989]; SEQ. ID. NO: 9), the human 52kd riboculeoprotein SS-A/Ro (Chan et al., J. Clin. Invest. 87, 68-76[1987]; Itoh et al., J. Clin. Invest. 87, 177-186 [1987]; A⁵² in ref. 1is P⁵² in ref. 2; SEQ. ID. NO: 10); human RING1 (Lovering, GBTRANSaccession number Z14000 [1992]; SEQ. ID. NO: 11), mouse T lymphocyteregulatory protein RPT-1 (Patarca et al., Proc. Natl. Acad. Sci. USA 85,2733-2737 [1988]; SEQ. ID.NO: 12), human regulatory protein RFP(Takahashi et al., Mol. Cell. Biol. 8, 1853-1856 [1988]; SEQ. ID. NO:13), and the product of the human proto-oncogen c-cbl (Blake et al.,Oncogene 6, 653-657 [1991]; SEQ. ID. NO: 14).

[0063] (B) Comparison of amino acid sequences containing TFIIIA-typezinc finger motifs. A region in TRAF2 comprising two contiguous repeatsof the consensus sequence C/H-X₂₋₄-C/H-X₂₋₁₅-C/H-X₂₋₄-C/H (Berg, J.Biol. Chem. 265, 6513-6516 [1990]) is aligned with similar zinc-bindingmotifs of the developmentally regulated DG17 gene product from D.discoideum (Driscoll & Williams, Mol. Cell. Biol. 7, 4482-4489 [1987];SEQ. ID. NO: 15), the transcription factor IIIA form X. laevis (Milleret al., EMBO J. 4, 1609-1614 [1985]; SEQ. ID. NO: 16), the Xenopus zincfinger proteins XLCOF14 and XFIN (Nietfeld et al., J. Mol. Biol. 208,639-659 [1989]; SEQ. ID. NO: 17; Ruiz i Altaba et al., EMBO J. 6,3065-3070 [1987]; SEQ. ID. NO: 18), the mouse ZFY1/2 and MFG2 geneproducts (Mardon & Page, Cell 56, 765-770 [1989]; SEQ. ID. NO: 19;Passananti et al., Proc. Natl. Acad. Sci. USA 86, 9421-9471 [1989]; SEQ.ID. NO: 20), and the RAD18 and UVS-2 proteins (see above; SEQ. ID. NOS:21 and 22).

[0064]FIG. 13. Homology between TRAF1 and TRAF2.

[0065] An optimized alignment of the protein sequences of TRAF1 andTRAF2 is shown. Identical amino acids are boxed. The C-terminal TRAFdomain (see text) comprises amino acids 180-409 of TRAF1 and 272-501 ofTRAF2.

[0066]FIG. 14. Hydropathy analysis of TRAF1 and TRAF2.

[0067] Hydropathy profiles of the amino acid sequences of TRAF1 (A) andTRAF2 (B) were obtained by the method of Kyte and Doolittle, J. Mol.Biol. 157, 105-132 (1982) using a window of twenty amino acids. Thenumbers under each plot indicate positions of the amino acids of therespective protein.

[0068]FIG. 15. Northern blot analysis of TRAF1 and TRAF2 mRNA.

[0069] (A) Northern blot analysis of TRAF1 and TRAF2 mRNA in CT6 cells.There is 3 μg of poly(A)⁺ RNA from CT6 cells per lane.

[0070] (B) Northern blot analysis of TRAF1 and TRAF2 mRNA in mousetissues. Mouse multiple tissue northern blots (Clontech) were hybridizedwith radiolabeled TRAF1 and TRAF2 probes as described in the text.

[0071]FIG. 16. Coprecipitation of GST-TRAF2 fusion protein in 293 cellextracts.

[0072] GST and GST-TRAF2 fusion protein beads were incubated withlysates from 293 and 293/TNF-R2 cells as described in the text.Reactions were analyzed by SDS-PAGE and Western blot analysis usinganti-human TNF-R1 monoclonal antibody 986 (0.5 μg/ml) and anti-humanTNF-R2 monoclonal antibody 1036 (0.5 μg/ml). An arrow indicates the75-80 kd hTNF-R2 band that is coprecipitated specifically with theGST-TRAF2 fusion protein. Molecular weight markers are indicated on theright in kd.

DETAILED DESCRIPTION OF THE INVENTION

[0073] A. Definitions

[0074] The phrases “factor,” “tumor necrosis factor receptor associatedfactor”, “TNF-R2 associated factor” and “TRAF” are used interchangeablyand refer to a native factor capable of specific association with theintracellular domain of a native TNF-R2, and functional derivatives ofsuch native factor. In the context of this definition the phrase“specific association” is used in the broadest sense, and includesdirect binding to a site or region within the intracellular domain of anative TNF-R2 of the human or of any animal species, and indirectassociation with a native TNF-R2 intracellular domain, mediated by afurther molecule, such as another TRAF. The phrase “native TRAF”designates a TRAF polypeptide as occurring in nature in any cell type ofany human or non-human animal species, with or without the initiatingmethionine, whether purified from native source, synthesized, producedby recombinant DNA technology or by any combination of these and/orother methods. Native TRAFs specifically include monomeric, homo- andheterodimeric and homo- and heterooligomeric forms of such naturallyoccurring polypeptides. The native TRAF polypeptides preferably share anovel sequence motif in the C-terminal portion of their amino acidsequences, and preferably are at least about 40%, more preferably atleast about 50%, most preferably at least about 55% homologous withinthis C-terminal “TRAF domain”. The “TRAF domain” encompasses about aminoacids 272 to 501 of the native mouse TRAF2 amino acid sequence, aboutamino acids 180 to 409 of the native mouse TRAF1 amino acid sequence,and homologous domains of other native TRAFs and their functionalderivatives.

[0075] The terms “native type 2 TNF receptor” and “native TNF-R2” areused interchangeably, and refer to any naturally occurring (native) type2 TNF receptor from any (human and non-human) animal species, with orwithout the initiating methionine and with or without a signal sequenceattached to the N-terminus, whether purified from native source,synthesized, produced by recombinant DNA technology or by anycombination of these and/or other methods.

[0076] The terms “native human type 2 TNF receptor” and “native humanTNF-R2”, which are used interchangeably, refer to a human TNF-R2 havingthe amino acid sequence disclosed in EP 418,014 (published Mar. 20,1991), with or without the initiating methionine and with or without asignal sequence attached to the N-terminus, whether purified from nativesource, synthesized, produced by recombinant DNA technology or by anycombination of these and/or other methods, and other naturally occurringhuman TNF-R2 variants, including soluble and variously glycosylatedforms of native full-length human TNF-R2, whether purified from naturalsources, synthetically produced in vitro or obtained by geneticmanipulation including methods of recombinant DNA technology.

[0077] A “functional derivative” of a native polypeptide is a compoundhaving a qualitative biological activity in common with the nativepolypeptide. Thus, a functional derivative of a native TRAF polypeptideis a compound that has a qualitative biological activity in common witha native TRAF. “Functional derivatives” include, but are not limited to,fragments of native polypeptides from any animal species (includinghumans), and derivatives of native (human and non-human) polypeptidesand their fragments, provided that they have a biological activity incommon with a respective native polypeptide. “Fragments” compriseregions within the sequence of a mature native polypeptide. The term“derivative” is used to define amino acid sequence and glycosylationvariants, and covalent modifications of a native polypeptide, whereasthe term “variant” refers to amino acid sequence and glycosylationvariants within this definition. Preferably, the functional derivativesare polypeptides which have at least about 65% amino acid sequenceidentity, more preferably about 75% amino acid sequence identity, evenmore preferably at least about 85% amino acid sequence identity, mostpreferably at least about 95% amino acid sequence identity with thesequence of a corresponding native polypeptide. Most preferably, thefunctional derivatives of a native TRAF polypeptide retain or mimic theregion or regions within the native polypeptide sequence that directlyparticipate in the association with the TNF-R2 intracellular domainand/or in homo- or heterodimerization. The phrase “functionalderivative” specifically includes peptides and small organic mleculeshaving a qualitative biological activity in common with a native TRAF.

[0078] The term “biological activity” in the context of the definitionof functional derivatives is defined as the possession of at least oneadhesive, regulatory or effector function qualitatively in common with anative polypeptide (e.g. TRAF). A preferred biological property of thefunctional derivatives of the native TRAF polypeptides herein is theirability to associate with the intracellular domain of a native TNF-R2(either by direct binding or via interaction with another TRAF), andthereby mediate or block a biological response signaled (exclusively orpartially) by the TNF-R2 with which they are associated.

[0079] “Identity” or “homology” with respect to a native polypeptide andits functional derivative is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical with theresidues of a corresponding native polypeptide, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent homology, and not considering any conservative substitutions aspart of the sequence identity. Neither N- or C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are well known in the art.

[0080] The TRAF polypeptides of the present invention specificallyinclude native murine TRAF-1 (SEQ. ID. NO: 2) and native murine TRAF-2(SEQ. ID. NO: 4) their homo- and heterodimeric and homo- andheterooligomeric forms, and their analogs in other mammalian species,such as rat, porcine, equine, cow, higher primates, and humans, and thefunctional derivatives of such native polypeptides. The functionalderivatives of a native TRAF-1 or native TRAF-2 receptor are preferablyencoded by DNAs capable, under stringent conditions, of hybridizing tothe complement of a DNA encoding a native TRAF polypeptide. Morepreferably, the functional derivatives share at least about 40% sequencehomology, more preferably at least about 50% sequence homology, evenmore preferably at least about 55% sequence homology, most preferably atleast about 60% sequence homology with any domain, and preferably withthe TNF-R2 binding domain(s) and/or the dimerization domain(s), of anative TRAF polypeptide. In a most preferred embodiment, a functionalderivative will share at least about 50% sequence homology with theC-terminal TRAF region of murine TRAF2, or are encoded by DNA capable ofhybridizing, under stringent conditions, with the complement of DNAencoding the TRAF region of murine TRAF2.

[0081] The “stringent conditions” are overnight incubation at 42° C. ina solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA.

[0082] The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-α-amino acids. The amino acids are identified by either thesingle-letter or three-letter designations: Asp D aspartic acid Ile Iisoleucine Thr T threonine Leu L leucine Ser S serine Tyr Y tyrosine GluE glutamic acid Phe F phenylalanine Pro P proline His H histidine Gly Gglycine Lys K lysine Ala A alanine Arg R arginine Cys C cysteine Trp Wtryptophan Val V valine Gln Q glutamine Met M methionine Asn Nasparagine

[0083] These amino acids may be classified according to the chemicalcomposition and properties of their side chains. They are broadlyclassified into two groups, charged and uncharged. Each of these groupsis divided into subgroups to classify the amino acids more accurately:

[0084] I. Charged Amino Acids

[0085] Acidic Residues: aspartic acid, glutamic acid

[0086] Basic Residues: lysine, arginine, histidine

[0087] II. Uncharged Amino Acids

[0088] Hydrophilic Residues: serine, threonine, asparagine, glutamine p1Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine

[0089] Non-polar Residues: cysteine, methionine, proline

[0090] Aromatic Residues: phenylalanine, tyrosine, tryptophan

[0091] The term “amino acid sequence variant” refers to molecules withsome differences in their amino acid sequences as compared to a nativeamino acid sequence.

[0092] Substitutional variants are those that have at least one aminoacid residue in a native sequence removed and a different amino acidinserted in its place at the same position. The substitutions may besingle, where only one amino acid in the molecule has been substituted,or they may be multiple, where two or more amino acids have beensubstituted in the same molecule.

[0093] Insertional variants are those with one or more amino acidsinserted immediately adjacent to an amino acid at a particular positionin a native sequence. Immediately adjacent to an amino acid meansconnected to either the α-carboxy or α-amino functional group of theamino acid.

[0094] Deletional variants are those with one or more amino acids in thenative amino acid sequence removed. Ordinarily, deletional variants willhave one or two amino acids deleted in a particular region of themolecule.

[0095] The term “glycosylation variant” is used to refer to aglycoprotein having a glycosylation profile different from that of anative counterpart or to glycosylated variants of a polypeptideunglycosylated in its native form(s). Glycosylation of polypeptides istypically either N-linked or O-linked. N-linked refers to the attachmentof the carbohydrate moiety to the side-chain of an asparagine residue.The tripeptide sequences, asparagine-X-serine andasparagine-X-threonine, wherein X is any amino acid except proline, arerecognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain. O-linked glycosylation refers tothe attachment of one of the sugars N-acetylgalactosamine, galactose, orxylose to a hydroxyamino acid, most commonly serine or threonine,although 5-hydroxyproline or 5-hydroxylysine may also be involved inO-linked glycosylation.

[0096] Antibodies (Abs) and immunoglobulins (Igs) are glycoproteinshaving the same structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

[0097] Native antibodies and immunoglobulins are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies between the heavy chainsof different immunoglobulin isotypes. Each heavy and light chain alsohas regularly spaced intrachain disulfide bridges. Each heavy chain hasat one end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one and (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains (Clothia etal., J. Mol. Biol. 186, 651-663 [1985]; Novotny and Haber, Proc. Natl.Acad. Sci. USA 82, 4592-4596 [1985]).

[0098] The term “variable” refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a β-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies (see Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, NationalInstitute of Health, Bethesda, Md. [1991]). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

[0099] Papain digestion of antibodies produces two identical antigenbinding fragments, called Fab fragments, each with a single antigenbinding site, and a residual “Fc” fragment, whose name reflects itsability to crystallize readily. Pepsin treatment yields an F(ab′)₂fragment that has two antigen combining sites and is still capable ofcross-linking antigen.

[0100] “Fv” is the minimum antibody fragment which contains a completeantigen recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

[0101] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other, chemical couplings of antibody fragments are also known.

[0102] The light chains of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda (λ), based on the amino acid sequences of theirconstant domains.

[0103] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, delta, epsilon, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

[0104] The term “antibody” is used in the broadest sense andspecifically covers single monoclonal antibodies (including agonist andantagonist antibodies), antibody compositions with polyepitopicspecificity, as well as antibody fragments (e.g., Fab, F(ab′)₂, and Fv),so long as they exhibit the desired biological activity.

[0105] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler & Milstein, Nature 256:495 (1975), ormay be made by recombinant DNA methods [see, e.g. U.S. Pat. No.4,816,567 (Cabilly et al.)].

[0106] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567(Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA 81,6851-6855 [1984]).

[0107] “Humanized” forms of non-human (e.g. murine) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibody may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see: Jones et al., Nature 321,522-525 [1986]; Reichmann et al., Nature 332, 323-329 [1988]; andPresta, Curr. Op. Struct. Biol. 2 593-596 [1992]).

[0108] In the context of the present invention the expressions “cell”,“cell line”, and “cell culture” are used interchangeably, and all suchdesignations include progeny. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological property, as screened for in the originally transformed cell,are included.

[0109] “Transformation” means introducing DNA into an organism so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integration.

[0110] “Transfection” refers to the taking up of an expression vector bya host cell whether or not any coding sequences are in fact expressed.

[0111] The terms “transformed host cell” and “transformed” refer to theintroduction of DNA into a cell. The cell is termed a “host cell”, andit may be a prokaryotic or a eukaryotic cell. Typical prokaryotic hostcells include various strains of E. coli. Typical eukaryotic host cellsare mammalian, such as Chinese hamster ovary cells or human embryonickidney 293 cells. The introduced DNA is usually in the form of a vectorcontaining an inserted piece of DNA. The introduced DNA sequence may befrom the same species as the host cell or a different species from thehost cell, or it may be a hybrid DNA sequence, containing some foreignand some homologous DNA.

[0112] The terms “replicable expression vector” and “expression vector”refer to a piece of DNA, usually double-stranded, which may haveinserted into it a piece of foreign DNA. Foreign DNA is defined asheterologous DNA, which is DNA not naturally found in the host cell. Thevector is used to transport the foreign or heterologous DNA into asuitable host cell. Once in the host cell, the vector can replicateindependently of the host chromosomal DNA, and several copies of thevector and its inserted (foreign) DNA may be generated. In addition, thevector contains the necessary elements that permit translating theforeign DNA into a polypeptide. Many molecules of the polypeptideencoded by the foreign DNA can thus be rapidly synthesized.

[0113] “Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods[such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as those described in EP 266,032, publishedMay 4, 1988, or via deoxynucleoside H-phosphanate intermediates asdescribed by Froehler et al., Nucl. Acids Res. 14, 5399 (1986)]. Theyare then purified on polyacrylamide gels.

[0114] B. Identification and Purification of TRAFs

[0115] The native TRAF polypeptides can be identified in and purifiedfrom certain tissues known to possess a type 2 TNF receptor (TNF-R2)mRNA and to express it at a detectable level. Thus, murine TRAF can, forexample, be obtained from the murine interleukin 2 (IL-2)-dependentcytotoxic T cell line CT6 (Ranges et al. J. Immunol. 142, 1203-1208[1989]). Murine TRAF1 can also be purified from spleen, lung and testis;whereas murine TRAF2 can be isolated and purified from an even largervariety of tissues, including heart, brain, spleen, lung, liver,skeletal muscle, kidney and testis (see FIG. 15b). In general, TRAFproteins are expected to be expressed in human tissues that are known toexpress TNF-R2, although not all of such tissues will express all TRAFs.Alternatively, TRAF polypeptides can be isolated from cell linestransfected with DNA encoding a native TNF-R2 or a TNF-R2 derivativecomprising intracellular domain sequences participating in theinteraction with TRAFs. Factors that are associated with theintracellular domain of a native TNF-R2 can be identified byimmunoprecipitation of the receptor or receptor derivative from cellsexpressing it. Immunoprecipitation in general consists of multipleordered steps, including lysing the cell with detergent if the TNF-R2 ismembrane-bound, binding of TNF-R2 to an anti-TNF-R2 antibody,precipitating the antibody complex, washing the precipitate, anddissociating TNF-R2 and any associated factor from the immune complex.The dissociated factor(s) can then be analyzed by electrophoreticmethods. In a preferred embodiment, radiolabeled TNF-R2 (or aderivative) is immunoprecipitated with protein A-agarose (OncogeneScience) or with protein A-Sepharose (Pharmacia). In this case, theTNF-R2/anti-TNF-R2 antibody immune complexes are precipitated byStaphylococcus aureus protein A bound to the agarose or Sepharose. Theimmunoprecipitate is then analyzed by autoradiography or byfluorography, depending on the actual radiolabel used. The TRAF proteins(which are characterized by their ability to associate with theintracellular domain of TNF-R2) will coprecipitate with the receptor orreceptor derivative, and can be further purified by methods known in theart, such as purification on an affinity column.

[0116] A large-scale purification scheme for purifying factors thatassociate with the intracellular domain of TNF-R2 takes advantage ofplasmid expression vectors that direct the synthesis of foreignpolypeptides in E. coli as fusions with the C terminus of glutathioneS-transferase (GST), as described by Smith, D. B. and Johnson, K. S.,Gene 67 31-40 (1988). The intracellular domain of TNF-R2 is expressed asa fusion protein with GST in E. coli recombinant host cells, and can bepurified from crude bacterial lysates by absorption onglutathione-agarose beads (Sigma). A cell lysate containing thefactor(s) to be purified is then applied to a GST-TNF-R2 fusion proteinaffinity column. Protein(s) bound to the column is/are eluted,precipitated and isolated by SDS-PAGE under reducing conditions, andvisualized by silver staining. GST gene fusion vectors (pGEX vectors) aswell as kits for cloning and expression of GST fusion systems arecommercially available from Pharmacia (see Pharmacia Catalog, 1994,pages 133; and 142-143).

[0117] Purified protein can be either sequenced directly by automatedEdman degradation with a model 470A Applied Biosystems gas phasesequencer equipped with a 120A PTH amino acid analyzer or sequencedafter digestion with various chemicals or enzymes. PTH amino acids wereintegrated using a ChromPerfect data system (Justice Innovations, PaloAlto, Calif.). Sequence interpretation can be performed on a VAX 11/785Digital Equipment Corporation computer as described by Henzel et al., J.Chromatography 404, 41 (1987). In some cases, eluates electrophoresed onSDS polyacrylamide gels are electrotransferred to a PVDF membrane(ProBlott, ABI, Foster City, Calif.) and stained with CoomassieBrilliant Blue R250 (Sigma). The specific protein is excised from theblot for N-terminal sequencing. To determine internal protein sequences,purified fractions obtained by reverse phase capillary HPLC aretypically dried under vacuum (SpeedVac), resuspended in appropriatebuffers, and digested with cyanogen bromide, and/or various proteases,such as trypsin, the lysine-specific enzyme Lys-C (Wako Chemicals,Richmond, Va.) or Asp-N (Boehringer Mannheim, Indianapolis, Ind.). Afterdigestion, the resultant peptides are sequenced as a mixture or areresolved by HPLC.

[0118] C. Recombinant Production of TRAF Polypeptides

[0119] Preferably, the TRAF polypeptides are prepared by standardrecombinant procedures by culturing cells transfected to express TRAFpolypeptide nucleic acid (typically by transforming the cells with anexpression vector) and recovering the polypeptide from the cells.However, it is envisioned that the TRAF polypeptides may be produced byhomologous recombination, or by recombinant production methods utilizingcontrol elements introduced into cells already containing DNA encodingan TRAF polypeptide. For example, a powerful promoter/enhancer element,a suppressor, or an exogenous transcription modulatory element may beinserted in the genome of the intended host cell in proximity andorientation sufficient to influence the transcription of DNA encodingthe desired TRAF polypeptide. The control element does not encode theTRAF polypeptide, rather the DNA is indigenous to the host cell genome.One next screens for cells making the polypeptide of this invention, orfor increased or decreased levels of expression, as desired.

[0120] Thus, the invention contemplates a method for producing a TRAFpolypeptide comprising inserting into the genome of a cell containingnucleic acid encoding a TRAF polypeptide a transcription modulatoryelement in sufficient proximity and orientation to the nucleic acidmolecule to influence transcription thereof, with an optional furtherstep of culturing the cell containing the transcription modulatoryelement and the nucleic acid molecule. The invention also contemplates ahost cell containing the indigenous TRAF polypeptide nucleic acidmolecule operably linked to exogenous control sequences recognized bythe host cell.

[0121] 1. Isolation of DNA Encoding the TRAF Polypeptides

[0122] For the purpose of the present invention, DNA encoding a TRAFpolypeptide can be obtained from cDNA libraries prepared from tissuebelieved to possess a type 2 TNF receptor (TNF-R2) mRNA and to expressit at a detectable level. For example, cDNA library can be constructedby obtaining polyadenylated mRNA from a cell line known to expressTNF-R2, and using the mRNA as a template to synthesize double strandedcDNA. Human and non-human cell lines suitable for this purpose have beenlisted hereinabove. It is noted, however, that TNF-R2 is known to beexpressed in a large variety of further tissues which can allpotentially serve as a source of TRAF cDNA, even though not all membersof the TRAF family will be expressed in all TNF-R2 expressing tissues.Alternatively, DNA encoding new TRAF polypeptides can be obtained fromcDNA libraries prepared from tissue known to express a previouslyidentified TRAF polypeptide at a detectable level. The TRAF polypeptidegenes can also be obtained from a genomic library, such as a humangenomic cosmid library.

[0123] Libraries, either cDNA or genomic, are screened with probesdesigned to identify the gene of interest or the protein encoded by it.For cDNA expression libraries, suitable probes include monoclonal andpolyclonal antibodies that recognize and specifically bind to a TRAFpolypeptide. For cDNA libraries, suitable probes include carefullyselected oligonucleotide probes (usually of about 20-80 bases in length)that encode known or suspected portions of a TRAF polypeptide from thesame or different species, and/or complementary or homologous cDNAs orfragments thereof that encode the same or a similar gene. Appropriateprobes for screening genomic DNA libraries include, without limitation,oligonucleotides, cDNAs, or fragments thereof that encode the same or asimilar gene, and/or homologous genomic DNAs or fragments thereof.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures as described in Chapters 10-12 ofSambrook et al., Molecular Cloning: A Laboratory Manual, New York, ColdSpring Harbor Laboratory Press, 1989).

[0124] A preferred method of practicing this invention is to usecarefully selected oligonucleotide sequences to screen cDNA librariesfrom various tissues. The oligonucleotide sequences selected as probesshould be sufficient in length and sufficiently unambiguous that falsepositives are minimized. The actual nucleotide sequence(s) is/areusually designed based on regions of a TRAF which have the least codonredundance. The oligonucleotides may be degenerate at one or morepositions. The use of degenerate oligonucleotides is of particularimportance where a library is screened from a species in whichpreferential codon usage is not known.

[0125] The oligonucleotide must be labeled such that it can be detectedupon hybridization to DNA in the library being screened. The preferredmethod of labeling is to use ATP (e.g., γ³²P) and polynucleotide kinaseto radiolabel the 5′ end of the oligonucleotide. However, other methodsmay be used to label the oligonucleotide, including, but not limited to,biotinylation or enzyme labeling.

[0126] cDNAs encoding TRAFs can also be identified and isolated by otherknown techniques of recombinant DNA technology, such as by directexpression cloning or by using the polymerase chain reaction (PCR) asdescribed in U.S. Pat. No. 4,683,195, issued Jul. 28, 1987, in section14 of Sambrook et al., Molecular Cloning: A Laboratory Manual, secondedition, Cold Spring Harbor Laboratory Press. New York, 1989 or inChapter 15 of Current Protocols in Molecular Biology, Ausubel et al.eds., Greene Publishing Associates and Wiley-lnterscience 1991. Thismethod requires the use of oligonucleotide probes that will hybridize toDNA encoding a TRAF.

[0127] According to a preferred method for practicing the invention, thecoding sequences for TRAF proteins can be identified in a recombinantcDNA library or a genomic DNA library based upon their ability tointeract with the intracellular domain of a TNF-R2. For this purpose onecan use the yeast genetic system described by Fields and co-workers(Fields and Song, Nature (London) 340, 245-246 [1989]; Chien et al.,Proc. Natl. Acad. Sci. USA 88, 9578-9582 [1991]) as disclosed by Chevrayand Nathans (Proc. Natl. Acad. Sci. USA 89, 5789-5793 [1991]). Manytranscriptional activators, such as yeast GAL4, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, while the other one functioning as the transcription activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GAL4, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

[0128] To directly isolate genes encoding proteins that associate withthe intracellular domain of TNF-R2, DNA encoding a TNF-R2 intracellulardomain or a fragment thereof is cloned into a vector containing DNAencoding the DNA-binding domain of GAL4. A plasmid cDNA library is thenconstructed by cloning double-stranded cDNA encoding a candidate factorin a vector comprising DNA encoding the GAL4 transcriptional activationdomain. Thereafter, yeast cells containing reporter genes arecotransformed with the TNF-R2-GAL4 DNA binding domain vector and withlibrary plasmid DNA. Typically, an S. cerevisiae cell containing tworeporter genes: lacZ(βgal) and His genes, serves as a host forcotransformation. Yeast transformants are selected by plating onsupplemented synthetic dextrose medium lacking tryptophan, leucine andhistidine, and protein-protein interactions are monitored by the yeastcolony filter β-galactosidase assay, essentially as described by Chevrayand Nathans, supra. Only colonies with protein-protein interaction willgrow on his plates, and are then analyzed for β-gal as a furthercontrol.

[0129] Once cDNA encoding a TRAF from one species has been isolated,cDNAs from other species can also be obtained by cross-specieshybridization. According to this approach, human or other mammalian cDNAor genomic libraries are probed by labeled oligonucleotide sequencesselected from known TRAF sequences (such as murine TRAF-1 and TRAF-2 asdisclosed in the present application) in accord with known criteria,among which is that the sequence should be sufficient in length andsufficiently unambiguous that false positives are minimized. Typically,a ³²P-labeled oligonucleotide having about 30 to 50 bases is sufficient,particularly if the oligonucleotide contains one or more codons formethionine or tryptophan. Isolated nucleic acid will be DNA that isidentified and separated from contaminant nucleic acid encoding otherpolypeptides from the source of nucleic acid.

[0130] Once the sequence is known, the gene encoding a particular TRAFpolypeptide can also be obtained by chemical synthesis, following one ofthe methods described in Engels and Uhlmann, Agnew. Chem. Int. Ed. Engl.28, 716 (1989). These methods include triester, phosphite,phosphoramidite and H-phosphonate methods, PCR and other autoprimermethods, and oligonucleotide syntheses on solid supports.

[0131] 2. Amino Acid Sequence Variants of a Native TRAF Proteins orFragments

[0132] Amino acid sequence variants of native TRAFs and TRAF fragmentsare prepared by methods known in the art by introducing appropriatenucleotide changes into a native or variant TRAF DNA, or by in vitrosynthesis of the desired polypeptide. There are two principal variablesin the construction of amino acid sequence variants: the location of themutation site and the nature of the mutation. With the exception ofnaturally-occurring alleles, which do not require the manipulation ofthe DNA sequence encoding the TRAF, the amino acid sequence variants ofTRAF are preferably constructed by mutating the DNA, either to arrive atan allele or an amino acid sequence variant that does not occur innature.

[0133] One group of the mutations will be created within the domain ordomains identified as being involved in the interaction with theintracellular domain of TNF-R2. TRAF variants mutated to enhance theirassociation (binding or indirect association) with TNF-R2 will be usefulas inhibitors of native TNF-R2/native TNF interaction. In addition, suchvariants will be useful in the diagnosis of pathological conditionsassociation with the overexpression of TNF-R2, and in the purificationof TNF-R2. A target for such mutations is the N-terminal RING fingerdomain of TRAF2 and related factors, as this domain is believed to beinvolved in the interaction with the intracellular domain of TNF-R2.

[0134] Another group of mutations will be performed within region(s)involved in interactions with other TNF-R2 associated factors. Thus,amino acid alterations within the homologous C-terminal domains (proteindimerization motif) of TRAF1, TRAF2 and other factors of the TRAF familycan enhance the ability of such factors to form stable dimers which arerequired for signaling through the TNF-R2 receptor.

[0135] Alternatively or in addition, amino acid alterations can be madeat sites that differ in TRAF proteins from various species, or in highlyconserved regions, depending on the goal to be achieved.

[0136] Sites at such locations will typically be modified in series,e.g. by (1) substituting first with conservative choices and then withmore radical selections depending upon the results achieved, (2)deleting the target residue or residues, or (3) inserting residues ofthe same or different class adjacent to the located site, orcombinations of options 1-3.

[0137] One helpful technique is called “alanine scanning” (Cunninghamand Wells, Science 244, 1081-1085 [1989]). Here, a residue or group oftarget residues is identified and substituted by alanine or polyalanine.Those domains demonstrating functional sensitivity to the alaninesubstitutions are then refined by introducing further or othersubstituents at or for the sites of alanine substitution.

[0138] After identifying the desired mutation(s), the gene encoding aTRAF variant can, for example, be obtained by chemical synthesis ashereinabove described.

[0139] More preferably, DNA encoding a TRAF amino acid sequence variantis prepared by site-directed mutagenesis of DNA that encodes an earlierprepared variant or a nonvariant version of the TRAF. Site-directed(site-specific) mutagenesis allows the production of TRAF variantsthrough the use of specific oligonucleotide sequences that encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 20 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered. In general, thetechniques of site-specific mutagenesis are well known in the art, asexemplified by publications such as, Edelman et al., DNA 2, 183 (1983).As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam (1981). Thisand other phage vectors are commercially available and their use is wellknown to those skilled in the art. A versatile and efficient procedurefor the construction of oligodeoxyribonucleotide directed site-specificmutations in DNA fragments using M13-derived vectors was published byZoller, M. J. and Smith, M., Nucleic Acids Res. 10, 6487-6500 [1982]).Also, plasmid vectors that contain a single-stranded phage origin ofreplication (Veira et al., Meth. Enzymol. 153, 3 [1987]) may be employedto obtain single-stranded DNA. Alternatively, nucleotide substitutionsare introduced by synthesizing the appropriate DNA fragment in vitro,and amplifying it by PCR procedures known in the art.

[0140] In general, site-specific mutagenesis herewith is performed byfirst obtaining a single-stranded vector that includes within itssequence a DNA sequence that encodes the relevant protein. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. USA 75, 5765 (1978). This primer is then annealedwith the single-stranded protein sequence-containing vector, andsubjected to DNA-polymerizing enzymes such as, E. coli polymerase IKlenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a heteroduplex is formed wherein one strand encodes theoriginal non-mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform appropriatehost cells such as JP101 cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.Thereafter, the mutated region may be removed and placed in anappropriate expression vector for protein production.

[0141] The PCR technique may also be used in creating amino acidsequence variants of a TRAF. When small amounts of template DNA are usedas starting material in a PCR, primers that differ slightly in sequencefrom the corresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template. For introduction of a mutation into a plasmid DNA,one of the primers is designed to overlap the position of the mutationand to contain the mutation; the sequence of the other primer must beidentical to a stretch of sequence of the opposite strand of theplasmid, but this sequence can be located anywhere along the plasmidDNA. It is preferred, however, that the sequence of the second primer islocated within 200 nucleotides from that of the first, such that in theend the entire amplified region of DNA bounded by the primers can beeasily sequenced. PCR amplification using a primer pair like the onejust described results in a population of DNA fragments that differ atthe position of the mutation specified by the primer, and possibly atother positions, as template copying is somewhat error-prone.

[0142] If the ratio of template to product material is extremely low,the vast majority of product DNA fragments incorporate the desiredmutation(s). This product material is used to replace the correspondingregion in the plasmid that served as PCR template using standard DNAtechnology. Mutations at separate positions can be introducedsimultaneously by either using a mutant second primer or performing asecond PCR with different mutant primers and ligating the two resultingPCR fragments simultaneously to the vector fragment in a three (or more)part ligation.

[0143] In a specific example of PCR mutagenesis, template plasmid DNA (1μg) is linearized by digestion with a restriction endonuclease that hasa unique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide triphosphates and isincluded in the GeneAmp^(R) kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μl. The reaction mixtureis overlayered with 35 μl mineral oil. The reaction is denatured for 5minutes at 100° C., placed briefly on ice, and then 1 μl Thermusaquaticus (Taq) DNA polymerase (5 units/l), purchased from Perkin-ElmerCetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineraloil layer. The reaction mixture is then inserted into a DNA ThermalCycler (purchased from Perkin-Elmer Cetus) programmed as follows:

[0144] 2 min. 55° C.,

[0145] 30 sec. 72° C., then 19 cycles of the following:

[0146] 30 sec. 94° C.,

[0147] 30 sec. 55° C., and

[0148] 30 sec. 72° C.

[0149] At the end of the program, the reaction vial is removed from thethermal cycler and the aqueous phase transferred to a new vial,extracted with phenol/chloroform (50:50 vol), and ethanol precipitated,and the DNA is recovered by standard procedures. This material issubsequently subjected to appropriate treatments for insertion into avector.

[0150] Another method for preparing variants, cassette mutagenesis, isbased on the technique described by Wells et al. [Gene 34, 315 (1985)].The starting material is the plasmid (or vector) comprising the TRAF DNAto be mutated. The codon(s) within the TRAF to be mutated areidentified. There must be a unique restriction endonuclease site on eachside of the identified mutation site(s). If no such restriction sitesexist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in the TRAF DNA. After the restriction sites havebeen introduced into the plasmid, the plasmid is cut at these sites tolinearize it. A double-stranded oligonucleotide encoding the sequence ofthe DNA between the restriction site but containing the desiredmutation(s) is synthesized using standard procedures. The two strandsare synthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 3′ and 5′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedTRAF DNA sequence.

[0151] Additionally, the so-called phagemid display method may be usefulin making amino acid sequence variants of native or variant TRAFs ortheir fragments. This method involves (a) constructing a replicableexpression vector comprising a first gene encoding an receptor to bemutated, a second gene encoding at least a portion of a natural orwild-type phage coat protein wherein the first and second genes areheterologous, and a transcription regulatory element operably linked tothe first and second genes, thereby forming a gene fusion encoding afusion protein; (b) mutating the vector at one or more selectedpositions within the first gene thereby forming a family of relatedplasmids; (c) transforming suitable host cells with the plasmids; (d)infecting the transformed host cells with a helper phage having a geneencoding the phage coat protein; (e) culturing the transformed infectedhost cells under conditions suitable for forming recombinant phagemidparticles containing at least a portion of the plasmid and capable oftransforming the host, the conditions adjusted so that no more than aminor amount of phagemid particles display more than one copy of thefusion protein on the surface of the particle; (f) contacting thephagemid particles with a suitable antigen so that at least a portion ofthe phagemid particles bind to the antigen; and (g) separating thephagemid particles that bind from those that do not. Steps (d) through(g) can be repeated one or more times. Preferably in this method theplasmid is under tight control of the transcription regulatory element,and the culturing conditions are adjusted so that the amount or numberof phagemid particles displaying more than one copy of the fusionprotein on the surface of the particle is less than about 1%. Also,preferably, the amount of phagemid particles displaying more than onecopy of the fusion protein is less than 10% of the amount of phagemidparticles displaying a single copy of the fusion protein. Mostpreferably, the amount is less than 20%. Typically in this method, theexpression vector will further contain a secretory signal sequence fusedto the DNA encoding each subunit of the polypeptide and thetranscription regulatory element will be a promoter system. Preferredpromoter systems are selected from lac Z, λ_(PL), tac, T7 polymerase,tryptophan, and alkaline phosphatase promoters and combinations thereof.Also, normally the method will employ a helper phage selected fromM13K07, M13R408, M13-VCS, and Phi X 174. The preferred helper phage isM13K07, and the preferred coat protein is the M13 Phage gene III coatprotein. The preferred host is E. coli, and protease-deficient strainsof E. coli.

[0152] Further details of the foregoing and similar mutagenesistechniques are found in general textbooks, such as, for example,Sambrook et al., supra, and Current Protocols in Molecular Biology,Ausubel et al. eds., supra.

[0153] Naturally-occurring amino acids are divided into groups based oncommon side chain properties:

[0154] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0155] (2) neutral hydrophobic: cys, ser, thr;

[0156] (3) acidic: asp, glu;

[0157] (4) basic: asn, gin, his, lys, arg;

[0158] (5) residues that influence chain orientation: gly, pro; and

[0159] (6) aromatic: trp, tyr, phe.

[0160] Conservative substitutions involve exchanging a member within onegroup for another member within the same group, whereas non-conservativesubstitutions will entail exchanging a member of one of these classesfor another. Variants obtained by non-conservative substitutions areexpected to result in significant changes in the biologicalproperties/function of the obtained variant, and may result in TRAFvariants which block TNF biological activities, especially if they areexclusively or primarily mediated by TNF-R2. Amino acid positions thatare conserved among various species and/or various receptors of the TRAFfamily are generally substituted in a relatively conservative manner ifthe goal is to retain biological function.

[0161] Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Deletions may be introduced into regions not directlyinvolved in the interaction with the TNF-R2 intracellular domain.

[0162] Amino acid insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.insertions within the TRAF protein amino acid sequence) may rangegenerally from about 1 to 10 residues, more preferably 1 to 5 residues,more preferably 1 to 3 residues. Examples of terminal insertions includethe TRAF polypeptides with an N-terminal methionyl residue, an artifactof its direct expression in bacterial recombinant cell culture, andfusion of a heterologous N-terminal signal sequence to the N-terminus ofthe TRAF molecule to facilitate the secretion of the mature TRAF fromrecombinant host cells. Such signal sequences will generally be obtainedfrom, and thus homologous to, the intended host cell species. Suitablesequences include STII or Ipp for E. coli, alpha factor for yeast, andviral signals such as herpes gD for mammalian cells.

[0163] Other insertional variants of the native TRAF molecules includethe fusion of the N- or C-terminus of the TRAF molecule to immunogenicpolypeptides, e.g. bacterial polypeptides such as beta-lactamase or anenzyme encoded by the E. coli trp locus, or yeast protein, andC-terminal fusions with proteins having a long half-life such asimmunoglobulin regions (preferably immunoglobulin constant regions),albumin, or ferritin, as described in WO 89/02922 published on Apr. 6,1989.

[0164] Since it is often difficult to predict in advance thecharacteristics of a variant TRAF, it will be appreciated that somescreening will be needed to select the optimum variant.

[0165] 3. Insertion of DNA into a Cloning Vehicle

[0166] Once the nucleic acid encoding a native or variant TRAF isavailable, it is generally ligated into a replicable expression vectorfor further cloning (amplification of the DNA), or for expression.

[0167] Expression and cloning vectors are well known in the art andcontain a nucleic acid sequence that enables the vector to replicate inone or more selected host cells. The selection of the appropriate vectorwill depend on 1) whether it is to be used for DNA amplification or forDNA expression, 2) the size of the DNA to be inserted into the vector,and 3) the host cell to be transformed with the vector. Each vectorcontains various components depending on its function (amplification ofDNA of expression of DNA) and the host cell for which it is compatible.The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

[0168] (i) Signal Sequence Component

[0169] In general, the signal sequence may be a component of the vector,or it may be a part of the TRAF molecule that is inserted into thevector. If the signal sequence is heterologous, it should be selectedsuch that it is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell.

[0170] As the TRAF molecules are intracellular proteins, they areunlikely to have a native signal sequence. Heterologous signal sequencessuitable for prokaryotic host cells are prokaryotic signal sequences,such as the alkaline phosphatase, penicillinase, lpp, or heat-stableenterotoxin II leaders. For yeast secretion the yeast invertase, alphafactor, or acid phosphatase leaders may be used. In mammalian cellexpression mammalian signal sequences are suitable.

[0171] (ii) Origin of Replication Component

[0172] Both expression and cloning vectors contain a nucleic acidsequence that enabled the vector to replicate in one or more selectedhost cells. Generally, in cloning vectors this sequence is one thatenables the vector to replicate independently of the host chromosomes,and includes origins of replication or autonomously replicatingsequences. Such sequence are well known for a variety of bacteria, yeastand viruses. The origin of replication from the well-known plasmidpBR322 is suitable for most gram negative bacteria, the 2μ plasmidorigin for yeast and various viral origins (SV40, polyoma, adenovirus,VSV or BPV) are useful for cloning vectors in mammalian cells. Originsof replication are not needed for mammalian expression vectors (the SV40origin may typically be used only because it contains the earlypromoter). Most expression vectors are “shuttle” vectors, i.e. they arecapable of replication in at least one class of organisms but can betransfected into another organism for expression. For example, a vectoris cloned in E. coli and then the same vector is transfected into yeastor mammalian cells for expression even though it is not capable ofreplicating independently of the host cell chromosome.

[0173] DNA is also cloned by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of the DNA encoding the desired heterologous polypeptide.However, the recovery of genomic DNA is more complex than that of anexogenously replicated vector because restriction enzyme digestion isrequired to excise the encoded polypeptide molecule.

[0174] (iii) Selection Gene Component

[0175] Expression and cloning vectors should contain a selection gene,also termed a selectable marker. This is a gene that encodes a proteinnecessary for the survival or growth of a host cell transformed with thevector. The presence of this gene ensures that any host cell whichdeletes the vector will not obtain an advantage in growth orreproduction over transformed hosts. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins, e.g.ampicillin, neomycin, methotrexate or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g. the gene encoding D-alanine racemase forbacilli.

[0176] One example of a selection scheme utilizes a drug to arrestgrowth of a host cell. Those cells that are successfully transformedwith a heterologous gene express a protein conferring drug resistanceand thus survive the selection regimen. Examples of such dominantselection use the drugs neomycin [Southern et al., J. Molec. Appl.Genet. 1, 327 (1982)], mycophenolic acid [Mulligan et al., Science 209,1422 (1980)], or hygromycin [Sudgen et al., Mol. Cel.. Biol. 5, 410-413(1985)]. The three examples given above employ bacterial genes undereukaryotic control to convey resistance to the appropriate drug G418 orneomycin (geneticin), xgpt (mycophenolic acid), or hygromycin,respectively.

[0177] Other examples of suitable selectable markers for mammalian cellsare dihydrofolate reductase (DHFR) or thymidine kinase. Such markersenable the identification of cells which were competent to take up thedesired nucleic acid. The mammalian cell transformants are placed underselection pressure which only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes the desired polypeptide. Amplification is the process bywhich genes in greater demand for the production of a protein criticalfor growth are reiterated in tandem within the chromosomes of successivegenerations of recombinant cells. Increased quantities of the desiredpolypeptide are synthesized from the amplified DNA.

[0178] For example, cells transformed with the DHFR selection gene arefirst identified by culturing all of the transformants in a culturemedium which lacks hypoxanthine, glycine, and thymidine. An appropriatehost cell in this case is the Chinese hamster ovary (CHO) cell linedeficient in DHFR activity, prepared and propagated as described byUrlaub and Chasin, Proc. Nat'l. Acad. Sci. USA 77, 4216 (1980). Aparticularly useful DHFR is a mutant DHFR that is highly resistant toMTX (EP 117,060). This selection agent can be used with any otherwisesuitable host, e.g. ATCC No. CCL61 CHO-K1, notwithstanding the presenceof endogenous DHFR. The DNA encoding DHFR and the desired polypeptide,respectively, then is amplified by exposure to an agent (methotrexate,or MTX) that inactivates the DHFR. One ensures that the cell requiresmore DHFR (and consequently amplifies all exogenous DNA) by selectingonly for cells that can grow in successive rounds of ever-greater MTXconcentration. Alternatively, hosts co-transformed with genes encodingthe desired polypeptide, wild-type DHFR, and another selectable markersuch as the neo gene can be identified using a selection agent for theselectable marker such as G418 and then selected and amplified usingmethotrexate in a wild-type host that contains endogenous DHFR. (Seealso U.S. Pat. No. 4,965,199).

[0179] A suitable selection gene for use in yeast is the trp1 genepresent in the yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature282:39; Kingsman et al., 1979, Gene 7:141; or Tschemper et al., 1980,Gene 10:157). The trp1 gene providesa selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, for example,ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics 85:12). The presence ofthe trpl lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2 deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

[0180] (iv) Promoter Component

[0181] Expression vectors, unlike cloning vectors, should contain apromoter which is recognized by the host organism and is operably linkedto the nucleic acid encoding the desired polypeptide. Promoters areuntranslated sequences located upstream from the start codon of astructural gene (generally within about 100 to 1000 bp) that control thetranscription and translation of nucleic acid under their control. Theytypically fall into two classes, inducible and constitutive. Induciblepromoters are promoters that initiate increased levels of transcriptionfrom DNA under their control in response to some change in cultureconditions, e.g. the presence or absence of a nutrient or a change intemperature. At this time a large number of promoters recognized by avariety of potential host cells are well known. These promoters areoperably linked to DNA encoding the desired polypeptide by removing themfrom their gene of origin by restriction enzyme digestion, followed byinsertion 5′ to the start codon for the polypeptide to be expressed.This is not to say that the genomic promoter for a TRAF polypeptide isnot usable. However, heterologous promoters generally will result ingreater transcription and higher yields of expressed TRAFs as comparedto the native TRAF promoters.

[0182] Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature 275:615(1978); and Goeddel et al., Nature 281:544 (1979)), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes. 8:4057 (1980) and EPO Appin. Publ. No. 36,776) and hybrid promoterssuch as the tac promoter (H. de Boer et al., Proc. Nat'l. Acad. Sci. USA80:21-25 (1983)). However, other known bacterial promoters are suitable.Their nucleotide sequences have been published, thereby enabling askilled worker operably to ligate them to DNA encoding TRAF (Siebenlistet al., Cell 20:269 (1980)) using linkers or adaptors to supply anyrequired restriction sites. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding a TRAF.

[0183] Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem.255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149 (1978); and Holland, Biochemistry 17:4900 (1978)),such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase,pyruvatedecarboxylase,phosphofructokinase,glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase.

[0184] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al., EP 73,657A. Yeast enhancers also areadvantageously used with yeast promoters.

[0185] Promoter sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

[0186] TRAF transcription from vectors in mammalian host cells may becontrolled by promoters obtained from the genomes of viruses such aspolyoma virus, fowipox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g. the actin promoter or an immunoglobulin promoter, fromheat shock promoters, and from the promoter normally associated with theTRAF sequence, provided such promoters are compatible with the host cellsystems.

[0187] The early and late promoters of the SV40 virus are convenientlyobtained as an SV40 restriction fragment which also contains the SV40viral origin of replication [Fiers et al., Nature 273:113 (1978),Mulligan and Berg, Science 209, 1422-1427 (1980); Pavlakis et al., Proc.Natl. Acad. Sci. USA 78, 7398-7402 (1981)]. The immediate early promoterof the human cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment [Greenaway et al., Gene 18, 355-360 (1982)]. Asystem for expressing DNA in mammalian hosts using the bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978. Seealso, Gray et al., Nature 295, 503-508 (1982) on expressing cDNAencoding human immune interferon in monkey cells; Reyes et al., Nature297, 598-601 (1982) on expressing human β-interferon cDNA in mouse cellsunder the control of a thymidine kinase promoter from herpes simplexvirus; Canaani and Berg, Proc. Natl. Acad. Sci. USA 79, 5166-5170 (1982)on expression of the human interferon β1 gene in cultured mouse andrabbit cells; and Gorman et al., Proc. Natl. Acad. Sci., USA 79,6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkeykidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells,HeLa cells, and mouse HIN-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

[0188] (v) Enhancer Element Component

[0189] Transcription of a DNA encoding the TRAFs of the presentinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp, that act on a promoter to increaseits transcription. Enhancers are relatively orientation and positionindependent having been found 5′ [Laimins et al., Proc. NatI. Acad. Sci.USA 78, 993 (1981)] and 3′ [Lasky et al., Mol Cel. Biol. 3, 1108 (1983)]to the transcription unit, within an intron [Banerji et al., Cell 33,729 (1983)] as well as within the coding sequence itself [Osborne etal., Mol. Cel. Biol. 4, 1293 (1984)]. Many enhancer sequences are nowknown from mammalian genes (globin, elastase, albumin, α-fetoprotein andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297, 17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theTRAF DNA, but is preferably located at a site 5′ from the promoter.

[0190] (vi) Transcription Termination Component

[0191] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the TRAF. The 3′untranslated regions also include transcription termination sites.

[0192] Construction of suitable vectors containing one or more of theabove listed components, the desired coding and control sequences,employs standard ligation techniques. Isolated plasmids or DNA fragmentsare cleaved, tailored, and religated in the form desired to generate theplasmids required.

[0193] For analysis to confirm correct sequences in plasmidsconstructed, the ligation mixtures are used to transform E. coli K12strain 294 (ATCC 31,446) and successful transformants selected byampicillin or tetracycline resistance where appropriate. Plasmids fromthe transformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res. 9, 309 (1981) or by the method of Maxam et al., Methods inEnzymology 65, 499 (1980).

[0194] Particularly useful in the practice of this invention areexpression vectors that provide for the transient expression inmammalian cells of DNA encoding a TRAF. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector. Transientsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded byclones DNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying analogs and variants of a TRAF.

[0195] Other methods, vectors, and host cells suitable for adaptation tothe synthesis of the TRAF polypeptides in recombinant vertebrate cellculture are described in Getting et al., Nature 293, 620-625 (1981);Mantel et al., Nature 281, 40-46 (1979); Levinson et al.; EP 117,060 andEP 117,058. A particularly useful plasmid for mammalian cell cultureexpression of the TRAF polypeptides is pRK5 (EP 307,247).

[0196] (vii) Construction and Analysis of Vectors

[0197] Construction of suitable vectors containing one or more of theabove listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

[0198] For analysis to confirm correct sequences in plasmidsconstructed, the ligation mixtures are used to transform E. coli K12strain 294 (ATCC 31,446) and successful transformants selected byampicillin or tetracycline resistance where appropriate. Plasmids fromthe transformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequences by the methods of Messing et al., NucleiAcids Res. 9, 309 (1981) or by the method of Maxam et al., Methods inEnzymology 65, 499 (1980).

[0199] (viii) Transient Expression Vectors

[0200] Particularly useful in the practice of this invention areexpression vectors that provide for the transient expression inmammalian cells of DNA encoding a TRAF polypeptide. In general,transient expression involves the use of an expression vector that isable to replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high level of a desired polypeptide encoded by theexpression vector. Sambrook et al., supra, pp. 16.17-16.22. Transientexpression systems, comprising a suitable expression vector and a hostcell, allow for the convenient positive screening of such polypeptidesfor desired biological or physiological properties. Thus transientexpression systems are particularly useful in the invention for purposesof identifying analogs and variants of native TRAF polypeptides withTRAF biological activity.

[0201] (ix) Suitable Exemplary Vertebrate Cell Vectors

[0202] Other methods, vectors, and host cells suitable for adaptation tothe synthesis of a TRAF polypeptide (including functional derivatives ofnative proteins) in -recombinant vertebrate cell culture are describedin Gething et al., Nature 293, 620-625 (1981); Mantei et al., Nature281, 40-46 (1979); Levinson et al., EP 117,060; and EP 117,058. Aparticularly useful plasmid for mammalian cell culture expression of aTRAF polypeptide is pRK5 (EP 307,247) or pSVI6B (PCT Publication No. WO91/08291).

[0203] D. Selection and Transformation of Host Cells

[0204] Suitable host cells for cloning or expressing the vectors hereinare the prokaryote, yeast or higher eukaryote cells described above.Suitable prokaryotes include gram negative or gram positive organisms,for example E. coli or bacilli. A preferred cloning host is E. coli 294(ATCC 31,446) although other gram negative or gram positive prokaryotessuch as E. coli B, E. coli X1776 (ATCC 31,537), E. coli W3110 (ATCC27,325), Pseudomonas species, or Serratia Marcesans are suitable.

[0205] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable hosts for vectors herein.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species and strains are commonly available and usefulherein, such as S. pombe [Beach and Nurse, Nature 290, 140 (1981)],Kluyveromvces lactis [Louvencourt et al., J. Bacteriol. 737 (1983)];yarrowia (EP 402,226); Pichia pastoris (EP 183,070), Trichoderma reesia(EP 244,234), Neurospora crassa [Case et al., Proc. Natl. Acad. Sci. USA76, 5259-5263 (1979)]; and Aspergillus hosts such as A. nidulans[Ballance et al., Biochem. Biophys. Res. Commun. 112, 284-289 (1983);Tilburn et al., Gene 26, 205-221 (1983); Yelton et al, Proc. Natl. Acad.Sci. USA 81, 1470-1474 (1984)] and A. niger [Kelly and Hynes, EMBO J. 4,475-479 (1985)].

[0206] Suitable host cells may also derive from multicellular organisms.Such host cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture, althoughcells from mammals such as humans are preferred. Examples ofinvertebrate cells include plants and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melangaster(fruitfly), and Bombyx mori host cells have been identified. See, e.g.Luckow et al., Bio/Technology 6, 47-55 (1988); Miller et al., in GeneticEngineering, Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing,1986), pp.277-279; and Maeda et al., Nature 315, 592-594 (1985). Avariety of such viral strains are publicly available, e.g. the L-1variant of Autographa californica NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

[0207] Plant cell cultures of cotton, corn, potato, soybean, petunia,tomato, and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the TRAF DNA. During incubation of the plant cell culture withA. tumefaciens, the DNA encoding a TRAF is transferred to the plant cellhost such that it is transfected, and will, under appropriateconditions, express the TRAF DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al., J. Mol. Appl. Gen. 1, 561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. SeeEP 321,196 published Jun. 21, 1989.

[0208] However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) is per sewell known. See Tissue Culture, Academic Press, Kruse and Patterson,editors (1973). Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney cell line [293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen. Virol. 36, 59 (1977)]; babyhamster kidney cells 9BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR [CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77 4216(1980)]; mouse sertolli cells [TM4, Mather, Biol. Reprod. 23, 243-251(1980)]; monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCCCCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells [Mather et al., Annals N.Y. Acad. Sci.383, 44068 (1982)]; MRC 5 cells; FS4 cells; and a human hepatoma cellline (Hep G2). Preferred host cells are human embryonic kidney 293 andChinese hamster ovary cells.

[0209] Particularly preferred host cells for the purpose of the presentinvention are vertebrate cells producing the TRAF polypeptides.

[0210] Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors and cultured inconventional nutrient media modified as is appropriate for inducingpromoters or selecting transformants containing amplified genes.

[0211] E. Culturing the Host Cells

[0212] Prokaryotes cells used to produced the TRAF polypeptides of thisinvention are cultured in suitable media as describe generally inSambrook et al., supra.

[0213] Mammalian cells can be cultured in a variety of media.Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium (DMEM, Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham and Wallace,Meth. Enzymol. 58, 44 (1979); Barnes and Sato, Anal. Biochem. 102, 255(1980), U.S. Pat. No. 4,767,704; U.S. Pat. No. 4,657,866; U.S. Pat. No.4,927,762; or U.S. Pat. No. 4,560,655; WO 90/03430; WO 87/00195 or U.S.Pat. No. Re. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug) trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH and the like, suitably arethose previously used with the host cell selected for cloning orexpression, as the case may be, and will be apparent to the ordinaryartisan.

[0214] The host cells referred to in this disclosure encompass cells inin vitro cell culture as well as cells that are within a host animal orplant.

[0215] It is further envisioned that the TRAF polypeptides of thisinvention may be produced by homologous recombination, or withrecombinant production methods utilizing control elements introducedinto cells already containing DNA encoding the particular TRAF.

[0216] F. Detecting Gene Amplification/Expression

[0217] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA 77, 5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as a site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to thesurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

[0218] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hse et al., Am. J. Clin. Pharm.75, 734-738 (1980).

[0219] Antibodies useful for immunohistochemical staining and/or assayof sample fluids may be either monoclonal or polyclonal, and may beprepared in any animal. Conveniently, the antibodies may be preparedagainst a native TRAF polypeptide, or against a synthetic peptide basedon the DNA sequence provided herein as described further hereinbelow.

[0220] G. Purification of the TRAF Polypeptides

[0221] The TRAF polypeptide is typically recovered from host celllysates.

[0222] When the TRAF polypeptide is expressed in a recombinant cellother than one of human origin, the TRAF is completely free of proteinsor polypeptides of human origin. However, it is necessary to purify theTRAF protein from recombinant cell proteins or polypeptides to obtainpreparations that are substantially homogenous as to the TRAF. As afirst step, the culture medium or lysate is centrifuged to removeparticulate cell debris. The membrane and soluble protein fractions arethen separated. The TRAF protein may then be purified from the solubleprotein fraction. The following procedures are exemplary of suitablepurification procedures: fractionation on immunoaffinity or ion-exchangecolumns; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; and protein A Sepharose columns to removecontaminants such as IgG. Specific purification procedures have beendescribed hereinabove.

[0223] TRAF functional derivatives in which residues have been deleted,inserted and/or substituted are recovered in the same fashion as thenative receptor chains, taking into account of any substantial changesin properties occasioned by the alteration. For example, fusion of theTRAF protein with another protein or polypeptide, e.g. a bacterial orviral antigen, facilitates purification; an immunoaffinity columncontaining antibody to the antigen can be used to absorb the fusion.Immunoaffinity columns such as a rabbit polyclonal anti-TRAF column canbe employed to absorb TRAF variant by binding to at least one remainingimmune epitope. A protease inhibitor, such as phenyl methyl sulfonylfluoride (PMSF) also may be useful to inhibit proteolytic degradationduring purification, and antibiotics may be included to prevent thegrowth of adventitious contaminants. The TRAF proteins of the presentinvention are conveniently purified by affinity chromatography, basedupon their ability to specifically associate with the intracellulardomain of a TNF-R2.

[0224] One skilled in the art will appreciate that purification methodssuitable for native TRAF may require modification to account for changesin the character of a native TRAF or its variants upon expression inrecombinant cell culture.

[0225] H. Covalent Modifications of TRAF Polypeptides

[0226] Covalent modifications of TRAF are included within the scopeherein. Such modifications are traditionally introduced by reactingtargeted amino acid residues of the TRAF with an organic derivatizingagent that is capable of reacting with selected sides or terminalresidues, or by harnessing mechanisms of post-translationalmodifications that function in selected recombinant host cells. Theresultant covalent derivatives are useful in programs directed atidentifying residues important for biological activity, for immunoassaysof the TRAF, or for the preparation of anti-TRAF receptor antibodies forimmunoaffinity purification of the recombinant. For example, completeinactivation of the biological activity of the protein after reactionwith ninhydrin would suggest that at least one arginyl or lysyl residueis critical for its activity, whereafter the individual residues whichwere modified under the conditions selected are identified by isolationof a peptide fragment containing the modified amino acid residue. Suchmodifications are within the ordinary skill in the art and are performedwithout undue experimentation.

[0227] Cysteinyl residues most commonly are reacted with α-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0228] Histidyl residues are derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1M sodium cacodylateat pH 6.0.

[0229] Lysinyl and amino terminal residues are reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

[0230] Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

[0231] The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

[0232] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiimides (R′—N═C═N−R′) such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

[0233] Glutaminyl and asparaginyl residues are frequently deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

[0234] Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl, threonyl or tyrosylresidues, methylation of the α-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86[1983]), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group. The molecules may further be covalentlylinked to nonproteinaceous polymers, e.g. polyethylene glycol,polypropylene glycol or polyoxyalkylenes, in the manner set forth inU.S. Ser. No. 07/275,296 or U.S. Pat. No. 4,640,835; U.S. Pat. No.4,496,689; U.S. Pat. No. 4,301,144; U.S. Pat. No. 4,670,417; U.S. Pat.No. 4,791,192 or U.S. Pat. No. 4,179,337.

[0235] Derivatization with bifunctional agents is useful for preparingintramolecular aggregates of the TRAF with polypeptides as well as forcross-linking the TRAF polypeptide to a water insoluble support matrixor surface for use in assays or affinity purification. In addition, astudy of interchain cross-links will provide direct information onconformational structure. Commonly used cross-linking agents include1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, homobifunctional imidoesters, andbifunctional maleimides. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water insoluble matrices such ascyanogen bromide activated carbohydrates and the systems reactivesubstrates described in U.S. Pat. Nos. 3,959,642; 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employedfor protein immobilization and cross-linking.

[0236] Certain post-translational modifications are the result of theaction of recombinant host cells on the expressed polypeptide.Glutaminyl and aspariginyl residues are frequently post-translationallydeamidated to the corresponding glutamyl and aspartyl residues.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

[0237] Other post-translational modifications include hydroxylation ofproline and lysine, phosphorylation of hydroxyl groups of seryl,threonyl or tyrosyl residues, methylation of the α-amino groups oflysine, arginine, and histidine side chains [T. E. Creighton, Proteins:Structure and Molecular Properties, W. H. Freeman & Co., San Francisco,pp. 79-86 (1983)].

[0238] Other derivatives comprise the novel peptides of this inventioncovalently bonded to a nonproteinaceous polymer. The nonproteinaceouspolymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymernot otherwise found in nature. However, polymers which exist in natureand are produced by recombinant or in vitro methods are useful, as arepolymers which are isolated from nature. Hydrophilic polyvinyl polymersfall within the scope of this invention, e.g. polyvinylalcohol andpolyvinylpyrrolidone. Particularly useful are polyvinylalkylene etherssuch a polyethylene glycol, polypropylene glycol.

[0239] The TRAF polypeptides may be linked to various nonproteinaceouspolymers, such as polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;U.S. Pat. No. 4,496,689; U.S. Pat. No. 4,301,144; U.S. Pat. No.4,670,417; U.S. Pat. No. 4,791,192 or U.S. Pat. No. 4,179,337.

[0240] The TRAF may be entrapped in microcapsules prepared, for example,by coacervation techniques or by interfacial polymerization, incolloidal drug delivery systems (e.g. liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th Edition, Osol, A., Ed. (1980).

[0241] I. Glycosylation Variants of the TRAFs

[0242] The native TRAFs are believed to be unglycosylated, however,variants having glycosylation are within the scope herein. For ease,changes in the glycosylation pattern of a native polypeptide are usuallymade at the DNA level, essentially using the techniques discussedhereinabove with respect to the amino acid sequence variants. Thus,glycosylation signals can be introduced into the DNA sequence of nativeTRAF polypeptides.

[0243] Chemical or enzymatic coupling of glycosides to the TRAFmolecules of the molecules of the present invention may also be used toadd carbohydrate substituents. These procedures are advantageous in thatthey do not require production of the polypeptide that is capable ofO-linked (or N-linked) glycosylation. Depending on the coupling modeused, the sugar(s) may be attached to (a) arginine and histidine, (b)free carboxyl groups, (c) free hydroxyl groups such as those ofcysteine, (d) free sulfhydryl groups such as those of serine, threonine,or hydroxyproline, (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 (published Sep. 11, 1987), and inAplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306.

[0244] J. Anti-TRAF Antibody Preparation

[0245] (i) Polyclonal antibodies

[0246] Polyclonal antibodies to a TRAF molecule generally are raised inanimals by multiple subcutaneous (sc) or intraperitoneal (ip) injectionsof the TRAF and an adjuvant. It may be useful to conjugate the TRAF or afragment containing the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g. keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for examplemaleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glytaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are different alkyl groups.

[0247] Animals are immunized against the immunogenic conjugates orderivatives by combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to {fraction (1/10)} the original amount ofconjugate in Freud's complete adjuvant by subcutaneous injection atmultiple sites. 7 to 14 days later the animals are bled and the serum isassayed for anti-TRAF antibody titer. Animals are boosted until thetiter plateaus. Preferably, the animal boosted with the conjugate of thesame TRAF, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

[0248] (ii) Monoclonal Antibodies

[0249] Monoclonal antibodies are obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.Thus, the modifier “monoclonal” indicates the character of the antibodyas not being a mixture of discrete antibodies.

[0250] For example, the anti-TRAF monoclonal antibodies of the inventionmay be made using the hybridoma method first described by Kohler &Milstein, Nature 256:495 (1975), or may be made by recombinant DNAmethods [Cabilly, et al., U.S. Pat. No. 4,816,567].

[0251] In the hybridoma method, a mouse or other appropriate hostanimal, such as hamster is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)].

[0252] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0253] Preferred myeloma cells are those that fuse efficiently, supportstable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 cells available from the American Type CultureCollection, Rockville, Md. USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies [Kozbor, J. Immunol. 133:3001 (1984);Brodeur, et al., Monoclonal Antibody Production Techniques andApplications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)].

[0254] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against TRAF.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

[0255] The binding affinity of the monoclonal antibody can, for example,be determined by the Scatchard analysis of Munson & Pollard, Anal.Biochem. 107:220 (1980).

[0256] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, Monoclonal Antibodies: Principles and Practice, pp.59-104(Academic Press, 1986). Suitable culture media for this purpose include,for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

[0257] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0258] DNA encoding the monoclonal antibodies of the invention isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Thehybridoma cells of the invention serve as a preferred source of suchDNA. Once isolated, the DNA may be placed into expression vectors, whichare then transfected into host cells such as simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also may be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murinesequences, Morrison, et al., Proc. Nat. Acad. Sci. 81, 6851 (1984), orby covalently joining to the immunoglobulin coding sequence all or partof the coding sequence for a non-immunoglobulin polypeptide. In thatmanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of an anti-TRAF monoclonal antibody herein.

[0259] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a TRAF andanother antigen-combining site having specificity for a differentantigen.

[0260] Chimeric or hybrid antibodies also may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

[0261] For diagnostic applications, the antibodies of the inventiontypically will be labeled with a detectable moiety. The detectablemoiety can be any one which is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ¹²⁵I, ³²P, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

[0262] Any method known in the art for separately conjugating theantibody to the detectable moiety may be employed, including thosemethods described by Hunter, et al., Nature 144:945 (1962); David, etal., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219(1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).

[0263] The antibodies of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press,Inc., 1987).

[0264] Competitive binding assays rely on the ability of a labeledstandard (which may be a TRAF polypeptide or an immunologically reactiveportion thereof) to compete with the test sample analyte (TRAF) forbinding with a limited amount of antibody. The amount of TRAF in thetest sample is inversely proportional to the amount of standard thatbecomes bound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies generally are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

[0265] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, of theprotein to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three part complex. David & Greene, U.S. Pat No. 4,376,110.The second antibody may itself be labeled with a detectable moiety(direct sandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

[0266] (iii) Humanized Antibodies

[0267] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature 332,323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0268] It is important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e. theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.For further details see U.S. application Ser. No. 07/934,373 filed Aug.21, 1992, which is a continuation-in-part of application Ser. No.07/715,272 filed Jun. 14, 1991.

[0269] Alternatively, it is now possible to produce transgenic animals(e.g. mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g. Jakobovits et al., Proc. Natl. Acad. Sci. USA 90, 2551-255(1993); Jakobovits et al., Nature 362, 255-258 (1993).

[0270] (iv) Bispecific Antibodies

[0271] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for a TRAF, the other one is for any other antigen, andpreferably for another receptor or receptor subunit. For example,bispecific antibodies specifically binding two different TRAFs, or a TNFreceptor (preferably TNF-R2) and a TRAF, are within the scope of thepresent invention.

[0272] Methods for making bispecific antibodies are known in the art.

[0273] Traditionally, the recombinant production of bispecificantibodies is based on the coexpression of two immunoglobulin heavychain-light chain pairs, where the two heavy chains have differentspecificities (Millstein and Cuello, Nature 305, 537-539 (1983)).Because of the random assortment of immunoglobulin heavy and lightchains, these hybridomas (quadromas) produce a potential mixture of 10different antibody molecules, of which only one has the correctbispecific structure. The purification of the correct molecule, which isusually done by affinity chromatography steps, is rather cumbersome, andthe product yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published May 13, 1993), and inTraunecker et al., EMBO 10, 3655-3659 (1991).

[0274] According to a different and more preferred approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, and secondand third constant regions of an immunoglobulin heavy chain (CH2 andCH3). It is preferred to have the first heavy chain constant region(CH1) containing the site necessary for light chain binding, present inat least one of the fusions. DNAs encoding the immunoglobulin heavychain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed incopending application Ser. No. 07/931,811 filed Aug. 17, 1992.

[0275] For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121, 210 (1986).

[0276] (v) Heteroconjugate Antibodies

[0277] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT applicationpublication Nos. WO 91/00360 and WO 92/200373; EP 03089).Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

[0278] L. Use of TRAF Molecules

[0279] Based upon their ability to specifically associate with theintracellular domain of TNF-R2, the TRAF molecules of the presentinvention can be used to purify TNF-R2, which, in turn, is useful in thetreatment of various pathological conditions associated with theexpression of TNF, such as endotoxic (septic) shock and rheumatoidarthritis (RA). The dose regimen effective in the treatment of these andother diseases can be determined by routine experimentation.

[0280] Therapeutic formulations of the present invention are preparedfor storage by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, Pluronics or PEG.

[0281] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

[0282] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution.

[0283] Therapeutic compositions herein generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

[0284] The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

[0285] Suitable examples of sustained release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et al., 1983,“Biopolymers” 22 (1): 547-556), poly (2-hydroxyethyl-methacrylate) (R.Langer, et al, 1981, “J. Biomed. Mater. Res.” 15: 167-277 and R. Langer,1982, Chem. Tech.” 12: 98-105), ethylene vinyl acetate (R. Langer etal., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988A). Sustainedrelease compositions also include liposomes. Liposomes containing amolecule within the scope of the present invention are prepared bymethods known per se: DE 3,218,121A; Epstein et al., 1985, “Proc. Natl.Acad. Sci. USA” 82: 3688-3692; Hwang et al., 1980, “Proc. Natl. Acad.Sci. USA” 77: 4030-4034; EP 52322A; EP 36676A; EP 88046A; EP 143949A; EP142641A; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324A. Ordinarily the liposomes are of thesmall (about 200-800 Angstroms) unilamelar type in which the lipidcontent is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal NT-4 therapy.

[0286] An effective amount of a molecule of the present invention to beemployed therapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 1 μg/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Typically, the clinician will administer a molecule ofthe present invention until a dosage is reached that provides therequired biological effect. The progress of this therapy is easilymonitored by conventional assays.

[0287] TRAF molecules may additionally be used to generate blocking(antagonist) or agonist anti-TRAF antibodies, which can be used to blockor mimic TNF biological activities mediated (exclusively or partially)by TNF-R2, or to purify other TRAF proteins having an epitope to whichthe antibodies bind. Methods for generating anti-TRAF antibodies havebeen described hereinabove. Other TRAF proteins may be identified andpurified, for example, by using the “two-hybrid” assay or its modifiedforms. Thus, factors (including native TRAF proteins and theirfunctional derivatives) that interact with the intracellular domain ofTNF-R2 primarily by dimerizing with a TRAF directly binding to thatdomain (e.g. TRAF2) can be identified by expressing nucleic acidmolecules encoding two fusion proteins in a single host cell transfectedwith nucleic acid encoding a TRAF capable of specific binding the TNF-R2intracellular domain. Specifically, nucleic acid molecules encoding afirst polypeptide comprising a fusion of an intracellular domainsequence of a native TNF-R2 to the DNA-binding domain of atranscriptional activator, and a second polypeptide comprising a fusionof a candidate polypeptide factor to the activation domain of atranscriptional activator are expressed in a single host celltransfected with nucleic acid encoding a polypeptide factor capable ofstrong specific binding to the intracellular domain of TNF-R2 (e.g.TRAF2), and with nucleic acid encoding a reporter gene. The associationof the candidate polypeptide with the intracellular domain of TNF-R2 orwith the polypeptide factor capable of binding to the intracellulardomain of TNF-R2is monitored by detecting the polypeptide encoded by thereporter gene.

[0288] TRAF molecules (including native TRAF polypeptides and functionalderivatives) can further be used in commercial screening assays toidentify further molecules that inhibit TNF signalling by disrupting theassociation of such TRAF molecules with TNF-R2. Such screening assaysmay, for example, be performed in a two-hybrid assay format as discussedhereinabove.

[0289] Further details of the invention will be apparent from thefollowing non-limiting examples.

EXAMPLE 1

[0290] Purification of TRAF-1

[0291] A. Cell Culture and Biological Reagents

[0292] The murine interleukin 2 (IL-2)-dependent cytotoxic T cell lineCT6 (Ranges et al. J. Immunol. 142, 1203-1208 [1989]) was cultured inRPMI 1640 media supplemented with 10-20 units recombinant human IL-2(Boehringer Mannheim), 10-15% fetal calf serum (Hyclone), 2 mML-glutamine, 10⁻⁵M β-mercaptoethanol, 100 units of penicillin per ml,and 100 μg of streptomycin per ml (GIBCO/BRL). The human T-cell lymphomaline Jurkat was obtained from the American Type Culture Collection(ATCC; Rockville, Md.) and maintained in RPMI 1640 media containing 10%fetal calf serum. The human embryonic kidney cell line 293 (ATCC CRL1573) and 293 cells overexpressing the hTNF-R2 (293/TNF-R2) weremaintained as described (Pennica et al., J. Biol. Chem. 267, 21172-21178[1992]). Recombinant hTNF and recombinant mTNF (specific activity of>10⁷ units/mg) were provided by the Genentech Manufacturing Group. Therabbit anti-human and anti-murine TNF-R2 antibodies have been describedpreviously (Pennica et al., supra; Tartaglia et al., J. Biol. Chem. 267,4304-4307 [1991]). Anti-human TNF-R1 monoclonal antibody 986 (IgG2aisotype) and anti-human TNF-R2 monoclonal antibodies 1036, 1035 and 1038(IgG2b, IgG2a and IgG2b isotypes, respectively) were produced asdescribed (Pennica et al., Biochemistry 31, 1134-1141 [1992]).

[0293] B. Mutational Analysis of the Intracellular Domain of hTNF-R2

[0294] It has been shown that the TNF induced proliferation of murineCT6 cells is mediated by the 75 kd TNF receptor (TNF-R2; Tartaglia etal., 1991, supra). In addition, TNF-R2 activates the transcriptionfactor NF-KB (Lenardo & Baltimore, Cell 58: 227-229 [1989]) and mediatesthe transcriptional induction of the granulocyte-macrophage colonystimulating factor (GM-CSF) gene (Miyatake et al, EMBO J. 4, 2561-2568[1985]; Stanley et al., EMBO J. 4, 2569-2573 [1985]) and the A20 zincfinger protein gene (Opipari et al., J. Biol. Chem. 265, 14705-14708[1990]) in CT6 cells (FIG. 1).

[0295] To identify sequences within the intracellular domain of thehTNF-R2 (hTNF-R2icd) that are required for TNF signaling a series ofmutant hTNF-R2 expression vectors was generated that encode receptorswith truncated intracellular domains. DNA fragments containingC-terminally truncated hTNR-R2icds were amplified from the full lengthexpression vector pRK-TNF-R2 (Tartaglia et al., Cell 73, 213-216 [1993])by PCR with Pfu DNA polymerase (Stratagene). PCR was run for 20 cycles(45 s at 95° C.; 60 s at 55° C.; 60 s at 72 C.) after an initial step of6 min at 95° C. A 0.5 kb DNA fragment encoding an intracellular domainwhich lacks amino acids 424-439 of the wild type hTNF-R2 was amplifiedusing the oligonucleotide primers 5′-CCTTGTGCCTGCAGAGAGAAG-3′ (SEQ. ID.NO: 23) and 5′-CTAGGTTAACTTTCGGTGCTCCCCAGCAGGGTCTC-3′ (SEQ. ID. NO: 24).The fragment was digested with PstI and HindII, gel purified, andre-cloned into the hTNF-R2 cDNA using the expression vector pRIS(Tartaglia & Goeddel, J. Biol. Chem. 267, 4304-4307 [1992];hTNF-R2(-16)). Similar mutant hTNF-R2 expression vectors were generatedthat encode receptors lacking amino acids 403-439(5′-CTAGGTTAACTGGAGAAGGGGACCTGCTCGTCCTT-3′ (SEQ. ID NO: 25);hTNF-R2(-37)), amino acids 381-439(5′-CTAGGTTAACTGCTGGCTTGGGAGGAGCACTGTGA-3′ (SEQ. ID NO: 26);hTNF-R2(-59)), amino acids 346-439 (5′-CTAGGTTAACTGCTCCCGGTGCTGGCCCGGGCCTC-3′ (SEQ. ID NO: 27); hTNF-R2(-94)) and amino acids 308-439(5′-CTAGGTTAACTGCACTGGCCGAGCTCTCCAGGGA-3′ (SEQ. ID NO: 28);hTNF-R2(-132)). A deletion of amino acids 304-345 of hTNF-R2 wasconstructed by partial digest of pRK-TNF-R2 with SacI and re-ligation ofthe vector (hTNF-R2(Δ304-345)). A deletion of the entire intracellulardomain of hTNF-R2 was constructed from pRK-TNF-R2 by replacement ofsequences between the PstI site adjacent to the transmembrane domain andthe C/al site with a double-stranded oligonucleotide(5′-GTGATGAGAATTCAT-3′ (SEQ. ID NO: 29) and 5′-CGATGAATTCTCATCACTGCA-3′) (SEQ. ID NO: 30) containing an in-frame stop codonimmediately following GIn²⁷³ (hTNF-R2(-166)). A mutation convertingSer³⁹³ into AIa was introduced into the hTNF-R2 cDNA by site-directedmutagenesis as described (Tartaglia et al., Cell 74, 845-853 [1993];hTNF-R2(S393A)). Verification of correctly modified cDNAs was determinedby double-strand sequencing using the Sequenase 2.0 Sequencing Kit (U.S. Biochemical).

[0296] The expression vectors encoding the intact and truncated hTNF-R2were introduced into CT6 cells by electroporation. 5×10⁶ cells in 0.3 mlRPMI 1640 media were cotransfected with 0.5 μg of ScaI-digested pRK.neo(Tartaglia & Goeddel, 1992, supra) and 20 μg of Scal-digested hTNF-R2expression vector using the Bio-Rad Gene Pulser with CapacitanceExtender (0.4 cm cuvette, 960 μF, 250 V). Electroporated cells wereresuspended in 50 ml media and after 2 days plated into 96-wellmicrotiter plates by limiting dilution in selective media containing 100μg/ml G418 (GIBCO/BRL). After three weeks, individual G418-resistantclones were picked and expanded. Clones that express the hTNF-R2 wereidentified by FACS analysis as described (Table 1; Pennica et al., J.Biol. Chem. 1992, supra).

[0297] Proliferation of CT6 clones expressing the full length andtruncated hTNF-R2 was measured by [³H]thymidine incorporation asdescribed (Tartaglia et al., Proc. Natl. Acad. Sci. USA 88, 9292-9296[1991]). NF-κB activation was analyzed by electrophoretic mobility shiftassay with nuclear extracts prepared from stimulated or unstimulated CT6cells as described (Schütze et al., Cell 71, 765-776 [1992]).

[0298] Table 1 shows that the transfected hTNF-R2 signals proliferationand NF-κB activation in CT6 cells. In addition, mutant human receptorswhich lack the C-terminal 16 amino acids or the internal 42 amino acids304-345 are still functional in mediating these activities. In contrast,mutant receptors which lack the C-terminal 37 amino acids or containfurther C-terminal deletions are defective in these assays. Theseresults indicate that a region of 78 amino acids within theintracellular domain of hTNF-R2 comprising amino acids 346-423 isrequired for mediating TNF signaling. This region contains a potentialprotein kinase C phosphorylation site (Ser³⁹³-Pro³⁹⁴-Lys³⁹⁵) which isconserved in the murine TNF-R2. However, a mutant hTNF-R2 containing Alainstead of Ser³⁹³ is biologically functional (Table 1) indicating thatthis phosphorylation site is not involved in TNF-R2 mediated signaling.

[0299] C. Identification of Factors that Associate with theIntracellular Domain of hTNF-R2

[0300] To identify factors that are associated with the intracellulardomain of hTNF-R2 immunoprecipitation of the receptor from lysates of[³⁵S]-labeled transfected CT6 cells was performed. 5×10⁶ CT6 cellsexpressing the wild type hTNF-R2 were washed twice with low glucoseDulbecco's modified Eagle's media without cysteine and methionine andincubated in fresh media for 30 min. The cells were seeded into a 100-mmplate in 5 ml media (without cysteine and methionine) containing[³⁵S]cysteine and [³⁵S]methionine (50 μCi of L-[³⁵S]-in vitro celllabeling mix/ml; Amersham). The cells were incubated for 4 h at 37° C.,stimulated for 10 min with 100 ng/ml hTNF, harvested, washed twice withcold PBS and lysed for 20 min at 4° C. in 1 ml of 0.1% NP40 lysis buffercontaining 50 mM HEPES pH 7.2, 250 mM NaCl, 10% Glycerol, 2 mM EDTA, 1mM PMSF, 1 μg/ml Benzamidine, 1 μg/ml Aprotinin, 1 μg/ml Leupeptin.Nuclear and cell debris were removed by centrifugation at 10,000×g for10 min at 4° C. The cell lysate was precleared for 1 h at 4° C. with 50μl Pansorbin (Calbiochem). The lysate was incubated for 8 h at 4° C.with 1 μg of each of the anti-hTNF-R2 monoclonal antibodies 1035 and1038 (directed against different epitopes of the extracellular domain ofthe hTNF-R2) that had been preabsorbed with 1 ml of unlabeled lysatefrom untransfected CT6 cells and collected with 15 μl of proteinA-agarose beads (Oncogene Science). The beads were washed extensivelywith lysis buffer, resuspended in SDS sample buffer and the supernatantelectrophoresed on a 4-12% or 8% Tris/glycine polyacrylamide gel. Thegel was fixed, incubated in Amplify (Amersham), dried, and exposed tofilm at −80° C.

[0301] Several bands in the range of 45-50 kd and one band ofapproximately 68 kd were specifically coprecipitated with theimmunoprecipitated hTNF-R2 in CT6 cells (FIG. 2a). The same result wasobtained when the hTNF-R2 was immunoprecipitated from unlabeled293/TNF-R2 cells followed by incubation with labeled lysate fromuntransfected CT6 cells (FIG. 2b). The pattern of bands coprecipitatedwith hTNF-R2 was identical regardless of whether the lysate was preparedfrom cells that had been stimulated with hTNF or left unstimulated,indicating that these proteins are constitutively associated with thehTNF-R2. This is similar to results observed for the tyrosine kinaseJAK2 which is associated with the intracellular domain of theerythropoietin receptor (Witthuhn et al., Cell 74, 227-236 [1993]). Inorder to establish a large scale purification procedure for factors thatassociate with the hTNF-R2icd, the intracellular domain of hTNF-R2 wasexpressed as a glutathione S-transferase (GST) fusion protein (Smith &Johnson, 1988, supra). The intracellular domain of hTNF-R2 was amplifiedfrom pRK-TNF-R2 by PCR with Pfu DNA polymerase as described above usingthe oligonucleotide primers 5′-GATCGGATCCAAAAAGAAGCC CTTGTGCCTGCA-3′(SEQ. ID NO: 31) and 5′-GCCTGGTTAACTGGGC-3 (SEQ. ID NO: 32). Theamplified 0.55 kb DNA fragment was blunt-ended, digested with BamHI andcloned into BamHI/SmaI-digested pGEX-2TK vector (Pharmacia;pGST-hTNF-R2icd). The pGST-hTNF-R2icd plasmid was transformed into aprotease deficient strain of E. coli K12 carrying the lacI^(q) gene onthe chromosome (Genentech), an overnight culture diluted 1:10 in freshLB-medium containing 100 μg/ml carbenicillin and grown at 37° C. for 2h. After induction with 0.1 mM IPTG, cells were grown for 1 h at 37° C.,pelleted and washed once with cold PBS. The cells were resuspended in1/100 culture volume of resuspension buffer containing 20 mM Tris-HCl pH7.5, 1 M NaCl, 5 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 μg/ml Benzamidine, 1μg/ml Aprotinin, 1 μg/ml Leupeptin. After sonication on ice, insolublematerial was removed by centrifugation at 10,000×g for 15 min at 4° C.Triton X-100 was added to 1% and the cell lysate incubated for 30 min atroom temperature on a rotator with 500 μl of a 50% slurry ofglutathione-agarose beads (sulphur linkage; Sigma) in PBS per 1 lculture volume. The beads were collected by brief centrifugation at500×g and washed extensively with resuspension buffer. An aliquot of thepurified GST-hTNF-R2icd fusion protein was analyzed by SDS-PAGE (FIG.3). Concentrations of 5-8 mg fusion protein/ml of swollen beads wereobtained routinely.

[0302] To prepare a covalently linked GST-hTNF-R2icd fusion proteinaffinity matrix, the fusion protein was eluted from glutathione-agarosebeads by competition with free glutathione using 3×30 min washes with 1bead volume of 250 mM Tris-HCl pH 8.0 containing 50 mM reducedglutathione (Sigma). The eluted fusion protein was dialyzed against 0.1M HEPES pH 7.2, 150 mM NaCl and covalently coupled to Affigel10/15 (2:1ratio; Bio-Rad) according to the instructions of the manufacturer.Fusion protein concentrations of up to 10 mg/ml of swollen beads wereobtained. Coprecipitation experiments with GST-hTNF-R2icd fusion proteinwere performed by incubating 3 μl fusion protein beads with 1 ml of celllysate prepared from [³⁵S]-labeled CT6 cells as described above. After 8h at 4° C. the fusion protein beads were extensively washed with lysisbuffer and analyzed by SDS-PAGE and autoradiography. A pattern of bandswas found to specifically coprecipitate with the GST-hTNF-R2icd fusionprotein either bound to glutathione-agarose beads or covalently coupledto Affigel10/15 (FIG. 4) that was very similar in size to the bandscoprecipitating with the immunoprecipitated hTNF-R2 (see FIG. 3). Thissuggests that the GST-hTNF-R2icd fusion protein expressed in E. colidoes associate with the same intracellular factors as the wild typehTNF-R2 in CT6 cells. Expression vectors were made that encodeGST-hTNF-R2icd fusion proteins with mutant intracellular domainsaccording to the mutational analysis described above. Using the samestrategy as for the wild type hTNF-R2icd DNA fragments encoding themutant-16, -37, -59 and Δ304-345 hTNF-R2 intracellular domains wereamplified by PCR and cloned into the pGEX-2TK vector. In addition, a0.14 kb DNA fragment was amplified using the oligonucleotide primers5′-GATCGGATCCGGAGACACAGATTCCA GCCCC-3′ (SEQ. ID NO: 53) and5′-GATCGAATTCTTAACTCTTCGGTGCTCCCCAGCAG-3′ (SEQ. ID NO: 54), digestedwith BamHI and EcoRI and cloned into pGEX-2TK. This DNA fragment encodesa peptide of 41 amino acids that correspond to amino acids 384-424 ofthe hTNF-R2icd. The fusion proteins were expressed, purified and assayedfor coprecipitating proteins as described above.

[0303] As shown in FIG. 5 the GST-hTNF-R2icd fusion proteins containingthe intracellular domains of the functional receptor mutantshTNF-R2(-16) and hTNF-R2(Δ304-345) coprecipitated the same bands as thefusion protein containing the wild type hTNF-R2icd. In contrast, theGST-hTNF-R2icd fusion proteins which contain the intracellular domainsof the inactive mutants hTNF-R2(-37) and hTNF-R2(-59) did notcoprecipitate these bands. This correlation between the biologicalactivity of hTNF-R2s with mutant intracellular domains and thecoprecipitation results obtained with the corresponding GST-hTNF-R2icdfusion proteins supports the observation that the wild typeGST-hTNF-R2icd fusion protein associates with the sameintracellularfactors as the immunoprecipitated hTNF-R2.

[0304] In addition, the GST-hTNF-R2icd(384-424) fusion protein was ableto coprecipitate the bands at 45-50 kd and 68 kd although to a weakerextent than the other fusion proteins (FIG. 5). The 41 amino acids ofthe hTNF-R2icd contained in this GST-fusion protein are comprised withinthe 78 amino acids region of the hTNF-R2icd that has been identified tobe required for mediating TNF signaling in CT6 cells (see above). Thissuggests that this short region of the hTNF-R2icd is sufficient tomediate the association of potential signaling molecules with thereceptor.

[0305] Competition coprecipitation experiments were performed in whichthe hTNF-R2 was immunoprecipitated from unlabeled 293/TNF-R2 cells andthen incubated with labeled CT6 cell lysate that had been preclearedwith 50 μl of GST-hTNF-R2icd fusion protein beads. Preincubation of theCT6 extracts with GST beads alone or GST-hTNF-R2icd(-37) andGST-hTNF-R2icd(-59) fusion protein beads had no effect on the pattern ofproteins coprecipitating with the immunoprecipitated hTNF-R2 (FIG. 6).However, if the cell lysate had been precleared with GST-hTNF-R2icd orGST-hTNF-R2icd(-16) fusion protein beads, these proteins did notcoprecipitate with the immunoprecipitated hTNF-R2 (FIG. 6), indicatingthat they had been depleted from the labeled CT6 cell extract by theGST-hTNF-R2icd fusion proteins. This result demonstrates that the wildtype GST-hTNF-R2icd fusion protein associates with the sameintracellular factors as the immunoprecipitated hTNF-R2. Consequently,this GST-fusion protein material can be used for large scalepurification of factors that are associated with the intracellulardomain the of hTNF-R2. Coprecipitation experiments of GST-hTNF-R2icdfusion beads with cell lysate prepared from [³⁵S] labeled human Jurkatcells revealed a pattern of coprecipitating proteins very similar insize to the pattern observed with murine CT6 lysates (FIG. 7). Thissuggests that the TNF-R2 associated factors are closely related betweenthe mouse and human species.

[0306] To investigate the subcellular localization of the hTNF-R2associated factors, cytoplasmic and membrane fractions from CT6 cellswere prepared essentially as described (Deutscher, Methods in Enzymol.182: Academic Press, San Diego [1990]). Briefly, [³⁵S] labeled CT6 cellswere washed once with cold PBS and once with isotonic salt buffercontaining 50 mM Tris HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1μg/ml Aprotinin and 1 μg/ml Leupeptin. Cells were resuspended in 5 mlisotonic salt buffer, and lysed in a glass douncer (Wheaton) with 20strokes using the ‘B’ pestle. Large cell debris were removed bycentrifugation at 750×g for 10 min at 4° C. and the supernatantsubjected to ultracentrifugation at 100,000×g for 30 min at 4° C. Thesupernatant which constitutes the cytoplasmic fraction was removed andthe pellet resuspended in 50 mM Tris HCl pH 7.4, 1 mM EDTA, 1 mM PMSF, 1μg/ml Aprotinin, 1 μg/ml Leupeptin. This crude membrane fraction waslayered on a cushion of 35% w/v sucrose in PBS and centrifuged at30,000×g for 45 min at 4° C. The purified cell membrane fraction at theinterface between the sucrose and the buffer phases was removedcarefully, concentrated by centrifugation at 100,000×g for 30 min at 4°C. and extracted with 0.1% NP40 lysis buffer for 30 min at 4° C. Thecell membrane lysate and the cytoplasmic fraction were used incoprecipitation experiments with GST-hTNF-R2 fusion protein beads asdescribed above.

[0307] The factors associating with the hTNF-R2icd were found to belocalized in the cytoplasmic cell fraction (FIG. 8). A small amountcould also be detected in the purified cell membrane fraction consistentwith the observation that these factors are constitutively associatedwith the hTNF-R2icd (see above).

[0308] D. Large Scale Purification

[0309] For large scale purification of hTNF-R2icd associated factors 60l of CT6 cells (3×10¹⁰ cells) were harvested and washed twice with coldPBS. All subsequent operations were carried out at 4° C. The cells werelysed by adding 120 ml of 0.1% NP40 lysis buffer containing 100 mM NaCland rocked gently for 30 min. Insoluble material was removed bycentrifugation for 10 min at 10,000×g. The supernatant was thencentrifuged at 100,000×g for 1 hr and dialyzed against lysis buffercontaining 500 mM NaCl. All subsequent purification steps were carriedout in lysis buffer containing 500 mM NaCl. The cell lysate was passedthrough a 15 ml glutathione-agarose GST-hTNF-R2icd(-37) fusion proteinpreabsorption column. The flow-through was applied to a 0.3 mlAffigel10/15 GST-hTNF-R2icd fusion protein affinity column. For control,the lysate was run through a similar Affigel10/15 GST-hTNF-R2icd(-37)fusion protein affinity column in parallel. After extensive washing,proteins bound to the resins were eluted with five column volumes ofImmunoPure Gentle Ag1Ab Elution Buffer (Pierce) containing, 0.1 M DTT,precipitated with Methanol/Chloroform and resuspended in SDS samplebuffer containing 5% SDS. One tenth of the material was separated bySDS-PAGE under reducing conditions and visualized by silver staining(FIG. 9). Protein bands that were specifically eluted from theGST-hTNF-R2icd fusion protein affinity column were observed atapproximately 45-50 kd and 68-70 kd.

[0310] The remaining purified material was separated by SDS-PAGE,electrophoretically transferred to PVDF sequencing membrane (Millipore)and proteins visualized by staining with R250. The protein band at 45-50kd (TNF Receptor Associated Factor 1 or TRAF1) was cut out and subjectedto amino acid sequence analysis by automated Edman degradation on anApplied Biosystems sequencer. Since the material proved to beN-terminally blocked, internal sequence information was obtained fromindividual peptides that were purified by reversed phase capillary HPLCafter protease digestion prior to sequence analysis. Two peptides thatwere obtained from trypsin and lysine C digestion, respectively, had thesequences APMALER and KHAYVK (SEQ>ID. NOS: 41 and 42).

EXAMPLE 2

[0311] Recombinant production of TRAF-1

[0312] The following degenerate oligonucleotides were designed based onthe sequences of the above peptides: BP50-1 sense,5′-GCNCCNATGGCNYTNGARC/AG (SEQ. ID. NOs: 33-35); BP50-1 antisense,5′-CT/GYTCNARNGCCATNGGNGC (SEQ. ID NOs: 36-38); BP50-11 sense,5′-AARCAYGCNTAY GTNAA (SEQ. ID NO: 39); BP50-11 antisense,5′-TTNACRTANGCRTGYTT (SEQ. ID NO: 40). 1 μg poly(A)⁺ mRNA isolated fromCT6 cells was oligo(dT)-primed and reverse transcribed using the cDNACycle Kit (Invitrogen) according to the instructions of themanufacturer. First-strand CT6 cDNA was subjected to PCR withcombinations of the degenerate oligonucleotides listed above using aCetus GeneAmp Kit and Perkin-Elmer Thermocycler. The PCR was run for 35cycles (45 s at 95° C.; 60 s at 55° C.; 150 s at 72° C.) after aninitial step of 6 min at 95° C. The PCR products were analyzed byelectrophoresis on a 1.6% agarose gel. The PCR reaction obtained withthe primer combination BP50-1 sense and BP50-11 antisense CoomaniiBrilliant Blue R-250 (Sigma) contained an amplified DNA fragment ofapproximately 0.75 kb. This fragment was gel-purified, subcloned intopBluescript KS (Stratagene), and sequenced.

[0313] A 0.65 kb PstI DNA fragment was isolated from the cloned PCRfragment and labeled with [λ-³²P]dCTP using the T7 Quick Prime Kit(Pharmacia). The labeled fragment was used to screen approximately 1×10⁶recombinant phage clones from a CT6 cDNA library that had beenconstructed in λgt22a using the Supercript Lambda System For cDNASynthesis And λ Cloning (GIBCO/BRL) according to the instructions of themanufacturer. Hybridization and washing of the filters were carried outunder high-stringency conditions according to standard protocols(Ausubel et al., Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley lnterscience, New York 1987). Fourpositive clones were plaque-purified by a secondary screen. The cDNAinserts of these phage clones were subcloned into pBluescript KS andsequenced on both strands (FIG. 10). The longest of the cDNAs was foundto be 2 kb. The other three cDNA clones represented truncated versionsof the 2 kb cDNA clone. The 2 kb cDNA clone contained an open readingframe encoding a protein of 409 amino acids (FIG. 10). Within thepredicted protein were the sequences APMALER (SEQ. ID. NO: 41) andKHAYVK (SEQ. ID. NO: 42), as well as the sequences PGSNLGS (SEQ. ID. NO:43) and KDDTMFLK (SEQ. ID. NO: 44) which correspond to two other peptidesequences obtained from protein sequence analysis. These results confirmthat the isolated 2 kb cDNA clone encodes the purified TRAF1.

[0314] To analyze similarities between TRAF1 and other known sequences,the TRAF1 sequence was searched against the Genentech protein database.No obvious similarity of significance between TRAF1 and any other knownprotein was found, indicating that TRAF1 is a novel molecule.

EXAMPLE 3

[0315] Identification and Cloning of TRAF2

[0316] To directly isolate genes coding for proteins that associate withthe intracellular domain of TNF-R2 the yeast two-hybrid system for thedetection of protein-protein interactions (Fields & Song, Nature 340,245-246 [1989]) was used. The intracellular domain of hTNF-R2 wasamplified from pRK-TNF-R2 by PCR with Pfu DNA polymerase as describedabove using the oligonucleotide primers 5′-TCGATCGTCGACCAAAAAGAAGCCCTCCTGCCTACAA-3′ (SEQ. ID NO: 45) and5′-CTAGAGATCTCAGG GGTCAGGCCACTTT-3′ (SEQ. ID. NO: 46). The amplified0.55 kb DNA fragment was digested with SalI and BglII, gel-purified andcloned into the GAL4 DNA-binding domain vector pPC97 (Chevray & Nathans,1992, supra; pPC97-hTNF-R2icd). Similar constructs were made containingthe GAL4 DNA-binding domain fused to the hTNF-R2icd(-16) (5′-CTAGAGATCTGTTAACTTTCGGTGCTCCCCAGCAGGGTCTC-3′ (SEQ. ID. NO: 47);pPC97-hTNF-R2icd (-16)), the hTNF-R2icd (-37)(5′-CTAGAGATCTGTTAACTGGAGAAGGGGACCTGCTCGTCC TT-3′ (SEQ. ID. NO: 48);pPC97-hTNF-R2icd(-37)), the hTNF-R2icd(-59) (5′-CTAGAGATCTGTTAACTGCTGGCTTGGGAGGAGCACTGTGA-3′ (SEQ. ID. NO: 49); pPC97-hTNF-R2icd(-59)) andthe intracellular domain of the murine TNF-R2(5′-TCGATCGTCGACCAAAAAGAAGCCCTCCTGCCT ACAA-3′ (SEQ. ID NO: 50)5′-CTAGAGATCTCAGGGGTCAGGCCACTTT-3′ (SEQ. ID. NO: 51); pPC97-mTNF-R2icd).

[0317] A plasmid cDNA library in the GAL4 transcriptional activationdomain vector pPC86 (Chevray & Nathans, 1992, supra) was constructedfrom SalI/NotI-adapted, double-stranded fetal liver stromal cell line7-4 cDNA (a gift of B. Bennett and W. Matthews) as described (Chevray &Nathans, 1992, supra). Plasmid DNA was isolated directly from 2×10⁶transformed E. coli DH10B (GIBCO/BRL) colonies. S. cerevisiae HF7c(Clontech) was sequentially transformed with pPC97-hTNF-R2icd and 250 μglibrary plasmid DNA as described in the Matchmaker Two-Hybrid System(Clontech). The final transformation mixture was plated onto 50 150-mmsynthetic dextrose agar plates lacking L-tryptophan, L-leucine,L-histidine and containing 20 mM 3-aminotriazole (Sigma). A total of2×10⁶ transformed colonies were plated. After 4 days at 30° C. 42surviving His⁺-colonies were obtained of which 15 were positive in afilter assay for b-galactosidase activity (Breeden & Nasmyth, Regulationof the Yeast HO gene. Cold Spring Harbor Symposia on QuantitativeBiology 50, 643-650, Cold Spring Harbor Laboratory Press, New York,1985). Yeast DNA was prepared (Hoffman and Winston, Gene 57, 267-272[1987]), transformed into E. Coli DH10B by electroporation and coloniescontaining the pPC86 library plasmid identified by restriction analysis.14 out of 15 cDNA inserts had a similar size of approximately 2.1 kb.Restriction analysis with Ddel revealed them to be independent cDNAclones derived from the same mRNA species.

[0318] Retransformation of three representative cDNA clones into HF7ccells with pPC97 and pPC97-hTNF-R2icd, respectively, confirmed that theencoded GAL4 activation domain fusion proteins do not interact with theGAL4 DNA-binding domain alone but only with the GAL4 DNA-bindinghTNF-R2icd fusion protein. The 2.1 kb cDNA insert of one representativeclone (pPC86Y17) was sequenced on both strands (FIG. 11). In addition,the 5′- and 3′-regions of 6 other independent cDNA clones were sequencedconfirming that they were derived from the same mRNA species. All cloneswere shown to be fused to the GAL4 DNA-binding domain in the samereading frame within 20 nucleotides of each other.

[0319] Two additional cDNA clones were isolated from a CT6 λ phage cDNAlibrary (see above) and 5 additional clones from a mouse liver λ phagecDNA library (Clontech) using a [³²P]-labeled 0.5 kb PstI DNA fragmentfrom the 5′-region of the pPC86Y17 cDNA insert as hybridization probe.None of these cDNA inserts extended the 5′-sequence of the pPC86 cDNAinserts. Furthermore, the size of the pPC86 cDNA inserts correspondsclosely to the size of the actual message as revealed by northern blotanalysis of CT6 mRNA (see below). These findings indicate that the cDNAinserts isolated with the two-hybrid system represent full length clonesand that the fusion to the GAL4 DNA-binding domain occurred in a veryshort 5′-untranslated region in-frame with the initiator ATG at position30 of the pPC86Y17 cDNA insert (see also below). The cDNA clones containan open reading frame encoding a protein of 501 amino acids (TNFReceptor Associated Factor 2 or TRAF2; FIG. 11).

[0320] A homology search of the TRAF2 sequence against the Genentechprotein database revealed that TRAF2 is a novel protein containing anN-terminal RING finger sequence motif (Freemont et al., Cell 64, 483-484[1991]; Haupt et al., Cell 65, 753-763 [1991]; Inoue et al., supra; FIG.12a). This sequence motif has been observed in the N-terminal domain ofa number of regulatory proteins and is thought to form two zinc-bindingfinger structures that appear to be involved in protein-DNA interactions(Freemont et al., supra; Haupt et al., supra; Reddy et al., TrendsBiochem. Sci. 17, 344-345 [1992]). Members of the RING finegr family areputative DNA-binding proteins, some of which are implicated intranscriptional regulation, DNA repair, and site-specific recombination(see FIG. 12a). In addition, the RING finger motif and otherzinc-binding sequence motifs have been discussed to be involved inprotein-protein interactions (Freemont et al., supra; Haupt et al.,supra; Berg, J. Biol. Chem. 265, 6513-6516 [1990]). The importance ofthis structural motif in TRAF2 is supported by the finding that all GAL4DNA-binding domain TRAF2 fusions isolated contain the completeN-terminus of TRAF2 (see above). This suggests that the N-terminal RINGfinger domain of TRAF2 is involved in the interaction with theintracellular domain of TNF-R2.

[0321] In addition, TRAF2 shares sequence similarity with the zincfinger motif of Xenopus TFIIIA-type zinc finger proteins (Miller et al.,EMBO J. 4, 1609-1614 [1985]; Berg, supra; FIG. 12b). TFIIIA-like zincfinger motifs have also been observed in the RING finger proteins RAD18and UVS-2 (FIG. 12).

[0322] No obvious similarity of significance between the C-terminaldomain of TRAF2 and any other known protein was found. A comparison ofthe sequences of TRAF1 and TRAF2 revealed that they share a high degreeof amino acid identify in their C-terminal domains (53% identity over230 amino acids; FIG. 13). Both proteins constitute members of a newfamily of proteins that contain a novel sequence homology motif, the“TRAF domain”. The less conserved N-terminal regions within the TRAFdomains of TRAF1 and TRAF2 can potentially form leucine zipper-likestructures (FIGS. 10, 11, 13). The leucine zipper is a α-helicalstructure originally found in a number of DNA-binding proteins thatcontain leucines occurring at intervals of every seventh amino acid(Landschulz et al., Science 240, 1759-1764 [1988]; Vinson et al.,Science 246, 911-916 [1989]). This structure mediates proteindimerization by intermolecular interaction of the leucine side-chains.Leucine zipper structures have also been predicted for two other RINGfinger family members, the SS-A/Ro ribonucleoprotein and the geneproduct of the c-cbl proto-oncogene (see FIG. 12). The N-terminaldomains of TRAF1 and TRAF2 are unrelated, especially with regard to theRING finger domain of TRAF2.

EXAMPLE 4

[0323] Functional Analysis of TRAF1 and TRAF2

[0324] Hydropathy profiles (Kyte & Doolittle, 1982, supra) of TRAF1 andTRAF2 (FIG. 14) suggest that they lack signal sequences as well asobvious transmembrane regions and are overall hydrophilic. They are thuslikely to represent intracellular proteins which is in accordance withthe cytoplasmic localization of TRAF1 as determined experimentally (seeabove).

[0325] Poly(A)⁺ mRNA was prepared from CT6 cells (Badley et al., CurrentOpinion in Structural Biology 3, 11-16, [1988]). Northern analysis(Sambrook et al., 1989, supra) using a radiolabeled TRAF2 hybridizationprobe as described above indicated that TRAF2 is expressed as a 2.1 kbmessage in CT6 cells (FIG. 15a). Similarly, TRAF1 is expressed in CT6cells as a 2 kb message (FIG. 15a).

[0326] To examine the tissue distribution of TRAF1 and TRAF2 mRNA, mousemultiple tissue Northern blots (Clontech) were hybridized withradiolabeled TRAF1 and TRAF2 probes according to the instructions of themanufacturer. TRAF2 is expressed constitutively in all mouse tissuesexamined (heart, brain, spleen, lung, liver, skeletal muscle, kidney andtestis; FIG. 15b). The highest expression level was observed in spleen.In contrast, TRAF1 displayed a tissue specific expression. TRAF1 mRNAcould only be detected in spleen, lung and testis (FIG. 15b).

[0327] Cotransformation of pPC86Y17 (pPC86TRAF2) into HF7c cells withthe above described GAL4 DNA-binding TNF-R2icd fusion constructs showedthat TRAF2 interacts with the wild type intracellular domains of boththe human and the murine TNF-R2 and with the intracellular domain of thebiologically active mutant hTNF-R2(-16) (Table 2). However it does notinteract with the GAL4 DNA-binding domain alone nor with theintracellular domains of the biologically inactive mutants hTNF-R2(-37)and hTNF-R2(-59). This is in agreement with the results obtained fromcoprecipitation experiments with wild type and mutant GST-hTNF-R2icdfusion proteins in CT6 cell extracts (see above).

[0328] An expression vector encoding a GST-TRAF2 fusion protein wasconstructed. The TRAF2 coding region was amplified from pPC86TRAF2 byPCR with Pfu DNA polymerase as described above using the oligonucleotideprimers 5′-GATCGGATCCTTGTGGTGTGTGGGGG TTGT (SEQ. ID. NO: 55) and5′-CCTGGCTGGCCTAATGT (SEQ. ID. NO: 56). The amplified 1.6 kb DNAfragment was blunt-ended using E. coli DNA polymerase 1, digested withBamHI and cloned into BamHI/SmaI-digested pGEX-2TK vector. The GST-TRAF2fusion protein was expressed in the presence of 1 mM ZnCL₂ and purifiedas described above. GST and GST-TRAF2 fusion protein beads wereincubated with lysates from 293 and 293/TNF-R2 cells, and analyzed bySDS-PAGE and Western blot analysis (Sambrook et al., “Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. [1989])using the ECL detection reagent (Amersham). Primary antibodies directedagainst the extracellular domains of hTNF-R2 and hTNF-R1 (as a control)were used at a concentration of 0.5 μg/ml and the secondary sheepanti-mouse horseradish peroxidase conjugate (Amersham) at a dilution of1:6000. As shown in FIG. 16, the GST-TRAF2 fusion protein coprecipitatesthe hTNF-R2 in 293 cell extracts, thus confirming the results obtainedfrom two hybrid analysis.

[0329] To test for possible homo- and heteromeric protein-proteininteractions between TRAF1, TRAF2 and the intracellular domain of TNF-R2the 2.1 kb cDNA insert of pPC86TRAF2 was excised by digestion with SalIand NotI and cloned into pPC97 (pPC97TRAF2). The TRAF1 coding and3′-untranslated region was amplified from the full length TRAF1 cDNAclone in pBluescript KS by PCR with Pfu DNA polymerase as describedabove using the T7 sequencing primer (Stratagene) and theoligonucleotide primer 5′-TCGATCGTCGACCGCCTCCAGCTCAGCCCCTGAT-3′ (SEQ.ID. NO: 52). The amplified 1.7 kb DNA fragment was digested with SalIand NotI, gel-purified and cloned into both pPC97 and pPC86 (pPC97TRAF1;pPC86TRAF1).

[0330] Cotransformation of pPC86TRAF1 into HF7c cells with the GAL4DNA-binding TNF-R2 fusion constructs encoding the wild type human andmurine intracellular domains indicated that the direct interactionbetween TRAF1 and the intracellular domain of TNF-R2 is weak (Table 2).However, cotransformation of pPC97TRAF1 and pPC86TRAF2 or pPC97TRAF2 andpPC86TRAF1 revealed that TRAF1 and TRAF2 interact with each other (Table2) suggesting that a heterodimeric complex of TRAF1 and TRAF2 isassociated with the intracellular domain of TNF-R2. Subsequently yeastvectors were constructed in which TRAF2 is expressed directly, i. e. notas a GAL4 fusion protein. pPC97TRAF2 was digested with HindIII and Sa/Ito release a 0.5 kb DNA fragment encoding the GAL4 DNA-binding domain,end-filled with Klenow enzyme, gel-purified, and re-ligated (pPCTRAF2).In addition, TRAF2 was amplified from pPC86TRAF2 with Pfu DNA polymeraseas described above using the oligonucleotide primers5′-GATCGACTCGAGATGCCCAAGAAGAAGCGGAAGGTGGC TGCAGCCAGTGTGACTTCCCCT (SEQ.ID. NO: 57) and 5′-CTCTGGCGAAGAAGTCC (SEQ. ID. NO: 58). The amplified2.1 kb DNA fragment was digested with XhoI, end-filled with Klenowenzyme, digested with NotI, gel-purified and cloned into pPC97 that hadbeen digested with HindIII, end-filled and digested with NotI(pPCTRAF2NLS). This expression vector encodes the simian virus 40 largetumor antigen nuclear localization signal(met-pro-lys-lys-lys-arg-lys-val; compare Chevray & Nathans, 1992) fusedto the N-terminus of TRAF2. Transformation of pPCTRAF2NLS but notpPCTRAF2 into HF7c cells harboring the plasmids pPC86TRAF1 andpPC97hTNF-R2icd or pPC97mTNF-R2icd complemented the histidine deficiencyof the host cells (Table 3). This result confirms that a heterodimericcomplex of TRAF1 and TRAF2 interacts with the intracellular domain ofTNF-R2. In this protein complex mainly TRAF2 contacts the receptordirectly potentially through interaction of its RING finger domain withthe C-terminal region of the intracellular domain comprising amino acids304-345 of the human TNF-R2 as suggested from mutational analysis andcoprecipitation experiments (see above). TRAF1 and TRAF2 can also formhomodimeric complexes as shown by cotransformation of pPC97TRAF1 andpPC86TRAF1 or pPC97TRAF2 and pPC86TRAF2 (Table 2). These results suggestthat the homologous C-terminal domains of TRAF1 and TRAF2 represent anovel protein dimerization motif. In analogy, the C-terminal domain ofthe RING finger protein COP1 from Arabidopsis thaliana contains a regionwith homology to the β subunit of trimeric G proteins that has beendiscussed to be involved in protein-protein recognition (Deng et al.,Cell 71, 791-801 [1992]).

[0331] Based on the tissue specific expression of TRAF1 (see above), theformation of a heteromeric complex between TRAF1 and TRAF2 can onlyoccur in certain mouse tissues such as spleen, lung and testis. Thisraises the possibility of other TRAF domain proteins as tissue specificdimerization partners for the constitutively expressed TRAF2. Suchtissue specific heterocomplexes with potentially different biologicalactivities could determine different TNF responses mediated by TNF-R2 invarious tissues.

[0332] To generate antibodies directed specifically against TRAF1 andTRAF2 the N-terminal domains of both proteins were expressed in E. colias 6xhis tag fusions using the QIAexpress system (Qiagen). A DNAfragment encoding amino acids 2-181 of TRAF1 was amplified from the fulllength cDNA clone in pBluescript KS by PCR with Pfu DNA polymerase asdescribed above using the oligonucleotide primers5′-GATCGGATCCGCCTCCAGCTCAGCCCCTGAT (SEQ. ID. NO: 59) and5′-GATCGGATCCAGCCAGCAGCTTCTCCTTCAC (SEQ. ID. NO: 60). The amplified 0.55kb DNA fragment was digested with BamHI and cloned into BamHI-digestedpQE12 vector (Qiagen). Transformants containing the correct orientationof the DNA insert in the expression vector were determined byrestriction analysis (pQETRAF1). Similarly, a DNA fragment encodingamino acids 1-162 of TRAF2 was amplified from pPC86TRAF2 using theoligonucleotide primers 5′-GATCGGATCCTTGTGGTGTGTGGGGGTTGT (SEQ. ID. NO:61) and 5′-GATCGGATCCGCTCAGG CTC TTTTGGGGCA (SEQ. ID. NO: 62), digestedwith BamHI and cloned into BamHI-digested pQE12 vector (pQETRAF2).Plasmids pQETRAF1 and pQETRAF2 were transformed into E. coli M15[pREP4](Qiagen). The cells were grown, induced, harvested, and the 6xhis tagTRAF1 and TRAF2 fusion proteins purified by Ni-NTA affinitychromatography (Qiagen) under denaturing conditions according to theinstructions of the manufacturer. The purified TRAF1 and TRAF2 fusionproteins were resolved on a 13% Tris/glycine polyacrylamide gel, stainedwith 0.05% Coomassie Brilliant Blue R-250 solution in water, and theappropriate bands excised. Gel slices containing 100-200 μg TRAF1 orTRAF2 fusion protein were used for the immunization of rabbits. TABLE 1Analysis of CT6 Clones Expressing Human TNF-R2 Mutants hTNF-R2[³H]Thymidine Expression Incorporation CT6 Clone (mean fluorescence)(fold stimulation) NF-κB Activation neo.26 157 0.9 − hR2.30 303 3.4 +hR2.31 350 4.7 + −16.25 464 10.7  + −16.31 465 5.1 + −37.4 478 1.1 −−37.20 439 1.0 − −59.1 276 1.3 − −59.23 374 1.0 − −94.5 515 0.7 − −94.6477 1.0 − −132.3 296 1.1 − −132.22 344 1.0 − −166.10 361 1.0 − −166.13318 1.0 − Δ304-345 469 nd + S393A.2 407 2.9 nd S393A.8 531 5.5 nd

[0333] Expression vectors encoding the intact and truncated hTNF-R2 weretransfected into CT6 cells. The expression levels of wild type or mutantreceptors of individual CT6 clones were analyzed by flow cytometry andvalues are expressed as mean fluorescence. For functional analysis twoindependent CT6 clones were examined for each hTNF-R2 mutant excepthTNF-R2(Δ304-345) which represents a pool of sorted cells. Proliferationwas measured by [³H]thymidine incorporation of cells that had beentreated for 24 hr with a 1:1000 dilution of anti-hTNF-R2 polyclonalantibody. Values are expressed as fold stimulation compared with cellsthat had been treated with an irrelevant antibody. Data shown are themeans of triplicate determinations. Standard deviations were generallyless than 5%. NF-KB activation was analyzed by electrophoretic mobilityshift assay with nuclear extracts prepared from cells that had beenstimulated for 20 min with a 1:500 dilution of anti-hTNF-R2 polyclonalantibody. A plus sign indicates the induction of NF-κB DNA-bindingactivity compared with nuclear extracts prepared from cells that hadbeen treated with an irrelevant antibody. All CT6 clones retained theability to induce proliferation and NF-κB activation through theendogenous murine TNF-R2 (data not shown). nd, not determined. TABLE 2Interaction between TRAF1, TRAF2 and the Intracellular Domain of TNF-R2Transformant Growth on trp- Growth on trp- DNA-binding domain hybridActivation-domain hybrid leu⁻ medium leu⁻ his⁻ medium GAL4(DB)GAL4(TA) + − GAL4(DB)-hTNF-R2icd GAL4(TA) + − GAL4(DB)-mTNF-R2icdGAL4(TA) + − GAL4(DB)-hTNF-R2icd(-16) GAL4(TA) + −GAL4(DB)-hTNF-R2icd(-37) GAL4(TA) + − GAL4(DB)-hTNF-R2icd(-59)GAL4(TA) + − GAL4(DB) GAL4(TA)-TRAF1 + − GAL4(DB) GAL4(TA)-TRAF2 + −GAL4(DB)-hTNF-R2icd GAL4(TA)-TRAF1 + −/+ GAL4(DB)-mTNF-R2icdGAL4(TA)-TRAF1 + −/+ GAL4(DB)-hTNF-R2icd GAL4(TA)-TRAF2 + +GAL4(DB)-mTNF-R2icd GAL4(TA)-TRAF2 + + GAL4(DB)-hTNF-R2icd(-16)GAL4(TA)-TRAF2 + + GAL4(DB)-hTNF-R2icd(-37) GAL4(TA)-TRAF2 + −GAL4(DB)-hTNF-R2icd(-59) GAL4(TA)-TRAF2 + − GAL4(DB)-TRAF1 GAL4(TA) + −GAL4(DB)-TRAF2 GAL4(TA) + − GAL4(DB)-TRAF1 GAL4(TA)-TRAF1 + +GAL4(DB)-TRAF1 GAL4(TA)-TRAF2 + + GAL4(DB)-TRAF2 GAL4(TA)-TRAF2 + +GAL4(DB)-TRAF2 GAL4(TA)-TRAF1 + +

[0334] HF7c cells were cotransformed with plasmids (see text) encodingvarious GAL4 DNA-binding domain (DB) and GAL4 transcriptional activationdomain (TA) fusion proteins as indicated. Aliquots of the sametransformation mixture were plated onto synthetic dextrose plateslacking trp and leu and plates lacking trp, leu, his and containing 20mM 3-aminotriazole. Plus signs indicate growth of transformed yeastcolonies on the respective plates. Very similar numbers of transformantsfrom the same transformation mixture (90-100%) were obtained on plateslacking trp, leu, his and on plates lacking trp and leu only. Minus/plussigns indicate that the number of transformants growing on plateslacking trp, leu, his was approximately 1-2% of the number of coloniesobtained from the same transformation mixture on plates lacking trp andleu only. Filter assays for β-galactosidase activity were performed oncolonies growing on plates lacking all three amino acids. All coloniesdeveloped a blue color (data not shown). TABLE 3 Interaction betweenTRAF1, TRAF2 and the Intracellular Domain of TNF-R2 TransformantActivation-domain Growth on trp- DNA-binding domain hybrid hybrid Directexpression leu⁻ his⁻ medium GAL4(DB) GAL4(TA)-TRAF1 − GAL4(DB)GAL4(TA)-TRAF1 NLS-TRAF2 − GAL4(DB) GAL4(TA)-TRAF1 TRAF2 −GAL4(DB)-hTNF-R2icd GAL4(TA)-TRAF1 −/+* GAL4(DB)-hTNF-R2icdGAL4(TA)-TRAF1 NLS-TRAF2 + GAL4(DB)-hTNF-R2icd GAL4(TA)-TRAF1 TRAF2 −/+*GAL4(DB)-mTNF-R2icd GAL4(TA)-TRAF1 −/+* GAL4(DB)-mTNF-R2icdGAL4(TA)-TRAF1 NLS-TRAF2 + GAL4(DB)-mTNF-R2icd GAL4(TA)-TRAF1 TRAF2 −/+*#plated onto synthetic dextrose plates lacking trp, leu, his andcontaining 20 mM 3-aminotriazole. Plus signs indicate growth oftransformed yeast colonies on the respective plates.

[0335] While the invention has necessarily been described in conjunctionwith preferred embodiments and specific working examples, one ofordinary skill, after reading the foregoing specification, will be ableto effect various changes, substitutions or equivalents, and alterationsto the subject matter set forth herein, without departing from thespirit and scope herein. Hence, the invention can be practices in waysother than those specifically described herein. All such modificationsare intended to be within the scope of the present invention.

[0336] All references cited herein and the references cited therein arehereby expressly incorporated by reference.

1 59 2088 base pairs Nucleic Acid Single Linear 1 CCCAGCCCGG TTCTCTGCCCCAAGGACGCT ACCGCCCAAT GCGAGCAGAA 50 GGCGGCGCAC AGATACAGAA AGTGAGGCTCAGACATATTG AAGACCGTGT 100 GACATAGGGT AGCCAAATGA CAGTGTGAGA AAGTGACATTTACTCAAGGC 150 CACCCAGATA TCCTGGAGGA CCCAGAACCC TGGAGATTCC CATCAGAAAG200 ACCTTCTGGC CACCTGAAAC CCCAAGATGG CCTCCAGCTC AGCCCCTGAT 250GAAAACGAGT TTCAATTTGG TTGCCCCCCT GCTCCCTGCC AGGACCCATC 300 GGAGCCCAGAGTTCTCTGCT GCACAGCCTG TCTCTCTGAG AACCTGAGAG 350 ATGATGAGGA TCGGATCTGTCCTAAATGCA GAGCAGACAA CCTCCATCCT 400 GTGAGCCCAG GAAGCCCTCT GACTCAGGAGAAGGTTCACT CTGATGTAGC 450 TGAGGCTGAA ATCATGTGCC CCTTTGCAGG TGTTGGCTGTTCCTTCAAGG 500 GGAGCCCACA ATCCATGCAG GAGCATGAGG CTACCTCCCA GTCCTCCCAC550 CTGTACCTGC TGCTGGCGGT CTTAAAGGAG TGGAAATCCT CACCAGGCTC 600CAACCTAGGG TCTGCACCCA TGGCACTGGA GCGGAACCTG TCAGAGCTGC 650 AGCTTCAGGCAGCTGTGGAA GCGACAGGGG ACCTGGAGGT AGACTGCTAC 700 CGGGCACCTT GCTGTGAGAGCCAGGAAGAA CTGGCCCTGC AGCACTTGGT 750 GAAGGAGAAG CTGCTGGCTC AGCTGGAGGAGAAGCTGCGT GTGTTTGCAA 800 ACATTGTTGC TGTCCTCAAC AAGGAAGTGG AGGCTTCCCACCTGGCACTG 850 GCCGCCTCCA TCCACCAGAG CCAGTTGGAC CGAGAGCACC TCCTGAGCTT900 GGAGCAGAGG GTGGTGGAAT TACAGCAAAC CCTGGCTCAA AAAGACCAGG 950TCCTGGGCAA GCTTGAGCAC AGTCTGCGAC TCATGGAGGA GGCATCCTTT 1000 GATGGTACTTTCCTGTGGAA GATCACCAAT GTCACCAAGC GGTGCCACGA 1050 GTCAGTGTGT GGCCGGACTGTCAGCCTCTT CTCTCCAGCT TTCTACACTG 1100 CCAAGTATGG TTACAAGTTG TGCCTGCGCTTGTACCTGAA CGGGGATGGC 1150 TCAGGCAAGA AGACCCACCT GTCCCTCTTC ATCGTGATCATGAGAGGAGA 1200 ATACGATGCT CTCCTGCCCT GGCCTTTCAG GAACAAGGTC ACCTTTATGC1250 TACTTGACCA GAACAACCGA GAGCATGCTA TTGATGCCTT CCGGCCTGAC 1300CTGAGCTCAG CCTCCTTCCA GCGGCCACAG AGTGAGACCA ACGTGGCCAG 1350 CGGCTGCCCGCTCTTCTTCC CCCTCAGCAA GCTGCAGTCA CCCAAGCACG 1400 CCTACGTCAA AGATGACACAATGTTCCTCA AATGCATTGT GGACACTAGT 1450 GCTTAGGGAT GGGGGGAGGG GGTGTCTCCTGACAGAACCA GCTTAGACTG 1500 GGGGACTTAG CTAGACAGCC AGGCCCTGCC TGCCCTTGGAGCCCACAGCC 1550 CACGACAAGG AGGAGCCAAG GCTGGCATGA CTTCAGCGCC ACAGCATGCT1600 GGTTATGGCT GATGTGAGGC TGGAGAAACG TGTGCGTACA GAGACAGAGT 1650GGAGGAGAAG ACAGAAGTGC TCTTTTCACA CAGACTACAC GACACCAGGA 1700 GGCCAGCATGCCAGCAGCTT CTGAATGTTG AGACCAGCCT AGATCAGGAT 1750 GAAAAGAGCC AGGCCTGAGGCTTGGACATT GAGCCAAGGC TATGGGGCCT 1800 AAGTGGAGGG GCACTCCTAC CAGGACATTCTCTCGAGGTC AGGGCATAAC 1850 TGGAAAAATG CCCCCATCTC TCTGTTCAGA CTCAAAACTAGAACCACAGG 1900 GCAGAAGGGT CAGACATTAA TGTGAATTTA ACCTGCCCTG GACTGAGTTC1950 CTATGTTAAC AGACACGCAA ACAGGTAAAC CCAGAAACTG CCCTGGGAAA 2000TGCTTTCTGG CTGCATCTGG AGATCTTTGA TGTTTTTACC GACAAAACAA 2050 ATAACAAAAGCCTTGAATTG CAAAAAAAAA AAAAAAAA 2088 409 amino acids Amino Acid Linear 2Met Ala Ser Ser Ser Ala Pro Asp Glu Asn Glu Phe Gln Phe Gly 1 5 10 15Cys Pro Pro Ala Pro Cys Gln Asp Pro Ser Glu Pro Arg Val Leu 20 25 30 CysCys Thr Ala Cys Leu Ser Glu Asn Leu Arg Asp Asp Glu Asp 35 40 45 Arg IleCys Pro Lys Cys Arg Ala Asp Asn Leu His Pro Val Ser 50 55 60 Pro Gly SerPro Leu Thr Gln Glu Lys Val His Ser Asp Val Ala 65 70 75 Glu Ala Glu IleMet Cys Pro Phe Ala Gly Val Gly Cys Ser Phe 80 85 90 Lys Gly Ser Pro GlnSer Met Gln Glu His Glu Ala Thr Ser Gln 95 100 105 Ser Ser His Leu TyrLeu Leu Leu Ala Val Leu Lys Glu Trp Lys 110 115 120 Ser Ser Pro Gly SerAsn Leu Gly Ser Ala Pro Met Ala Leu Glu 125 130 135 Arg Asn Leu Ser GluLeu Gln Leu Gln Ala Ala Val Glu Ala Thr 140 145 150 Gly Asp Leu Glu ValAsp Cys Tyr Arg Ala Pro Cys Cys Glu Ser 155 160 165 Gln Glu Glu Leu AlaLeu Gln His Leu Val Lys Glu Lys Leu Leu 170 175 180 Ala Gln Leu Glu GluLys Leu Arg Val Phe Ala Asn Ile Val Ala 185 190 195 Val Leu Asn Lys GluVal Glu Ala Ser His Leu Ala Leu Ala Ala 200 205 210 Ser Ile His Gln SerGln Leu Asp Arg Glu His Leu Leu Ser Leu 215 220 225 Glu Gln Arg Val ValGlu Leu Gln Gln Thr Leu Ala Gln Lys Asp 230 235 240 Gln Val Leu Gly LysLeu Glu His Ser Leu Arg Leu Met Glu Glu 245 250 255 Ala Ser Phe Asp GlyThr Phe Leu Trp Lys Ile Thr Asn Val Thr 260 265 270 Lys Arg Cys His GluSer Val Cys Gly Arg Thr Val Ser Leu Phe 275 280 285 Ser Pro Ala Phe TyrThr Ala Lys Tyr Gly Tyr Lys Leu Cys Leu 290 295 300 Arg Leu Tyr Leu AsnGly Asp Gly Ser Gly Lys Lys Thr His Leu 305 310 315 Ser Leu Phe Ile ValIle Met Arg Gly Glu Tyr Asp Ala Leu Leu 320 325 330 Pro Trp Pro Phe ArgAsn Lys Val Thr Phe Met Leu Leu Asp Gln 335 340 345 Asn Asn Arg Glu HisAla Ile Asp Ala Phe Arg Pro Asp Leu Ser 350 355 360 Ser Ala Ser Phe GlnArg Pro Gln Ser Glu Thr Asn Val Ala Ser 365 370 375 Gly Cys Pro Leu PhePhe Pro Leu Ser Lys Leu Gln Ser Pro Lys 380 385 390 His Ala Tyr Val LysAsp Asp Thr Met Phe Leu Lys Cys Ile Val 395 400 405 Asp Thr Ser Ala 4092121 base pairs Nucleic Acid Single Linear 3 GCGCGAAGAC CGTTGGGGCTTTGTGGTGTG TGGGGGTTGT AACTCACATG 50 GCTGCAGCCA GTGTGACTTC CCCTGGCTCCCTAGAACTGC TACAGCCTGG 100 CTTCTCCAAG ACCCTCCTGG GGACCAGGTT AGAAGCCAAGTACCTCTGTT 150 CAGCCTGCAA AAACATCCTG CGGAGGCCTT TCCAGGCCCA GTGTGGGCAC200 CGCTACTGCT CCTTCTGCCT GACCAGCATC CTCAGCTCTG GGCCCCAGAA 250CTGTGCTGCC TGTGTCTATG AAGGCCTGTA TGAAGAAGGC ATTTCTATTT 300 TAGAGAGTAGTTCGGCCTTT CCAGATAACG CTGCCCGCAG AGAGGTGGAG 350 AGCCTGCCAG CTGTCTGTCCCAATGATGGA TGCACTTGGA AGGGGACCTT 400 GAAAGAATAC GAGAGCTGCC ACGAAGGACTTTGCCCATTC CTGCTGACGG 450 AGTGTCCTGC ATGTAAAGGC CTGGTCCGCC TCAGCGAGAAGGAGCACCAC 500 ACTGAGCAGG AATGCCCCAA AAGGAGCCTG AGCTGCCAGC ACTGCAGAGC550 ACCCTGTAGC CACGTGGACC TGGAGGTACA CTATGAGGTC TGCCCCAAGT 600TTCCCTTAAC CTGTGATGGC TGTGGCAAGA AGAAGATCCC TCGGGAGACG 650 TTTCAGGACCATGTTAGAGC ATGCAGCAAA TGCCGGGTTC TCTGCAGATT 700 CCACACCGTT GGCTGTTCAGAGATGGTGGA GACTGAGAAC CTGCAGGATC 750 ATGAGCTGCA GCGGCTACGG GAACACCTAGCCCTACTGCT GAGCTCATTC 800 TTGGAGGCCC AAGCCTCTCC AGGAACCTTG AACCAGGTGGGGCCAGAGCT 850 ACTCCAGCGG TGCCAGATTT TGGAGCAGAA GATAGCAACC TTTGAGAACA900 TTGTCTGCGT CTTGAACCGT GAAGTAGAGA GGGTAGCAGT GACTGCAGAG 950GCTTGTAGCC GGCAGCACCG GCTAGACCAG GACAAGATTG AGGCCCTGAG 1000 TAACAAGGTGCAACAGCTGG AGAGGAGCAT CGGCCTCAAG GACCTGGCCA 1050 TGGCTGACCT GGAGCAGAAGGTCTCCGAGT TGGAAGTATC CACCTATGAT 1100 GGGGTCTTCA TCTGGAAGAT CTCTGACTTCACCAGAAAGC GTCAGGAAGC 1150 CGTAGCTGGC CGGACACCAG CTATCTTCTC CCCAGCCTTCTACACAAGCA 1200 GATATGGCTA CAAGATGTGT CTACGAGTCT ACTTGAATGG CGACGGCACT1250 GGGCGGGGAA CTCATCTGTC TCTCTTCTTC GTGGTGATGA AAGGCCCCAA 1300TGATGCTCTG TTGCAGTGGC CTTTTAATCA GAAGGTAACA TTGATGTTGC 1350 TGGACCATAACAACCGGGAG CATGTGATCG ACGCATTCAG GCCCGATGTA 1400 ACCTCGTCCT CCTTCCAGAGGCCTGTCAGT GACATGAACA TCGCCAGTGG 1450 CTGCCCCCTC TTCTGCCCTG TGTCCAAGATGGAGGCCAAG AATTCCTATG 1500 TGCGGGATGA TGCGATCTTC ATCAAAGCTA TTGTGGACCTAACAGGACTC 1550 TAGCCACCCC TGCTAAGAAT AGCAGCTCAG TGAGGAGCTG TCACATTAGG1600 CCAGCCAGGC CCTGCCACAC ACGGGTGGGC AGGCTTGGTG TAAATGCTGG 1650GGAGGGCCTC AGCCTAGAGC CAATCACCAT CACACAGAAA GGCAGGAAGA 1700 AGCCTCCAGTTGGCCTTCAG CTGGCAAACT GAGTTGGACG GTCCACTGAG 1750 CTCAAGGGCC TGGTGGAGCCCGCTGGGGAG CTTCTCAGCT TTCCAATAGG 1800 AAAGCTCCTG CTGTCTCCTC TGTCTGGGGAAGGGAGAGAC CTGTAGGTGG 1850 GTGCTCAGAA AGGGCCTCTC CAGAGAGAGT CTCAAGAGCTGCAGCAGGAG 1900 CAAAGTGACT GGCCTTCCCC ACCCCATCCT TTGGAAAAGA GGTAGCGGCT1950 ACACAGGAGA AGGCATGCGC CTGCAGGGTG TAGCCCAAGA GAGAAGCTCT 2000CTGAGACATA GGCCCTCACT GGAGAAGGGC CTGCCTGGGC TGCACAGCCT 2050 TGCCAGGTGGCCTGTATGGG GGAGAAGTGA TTAAATGTTG AGATGTCACA 2100 CGACAAAAAA AAAAAAAAAA A2121 501 amino acids Amino Acid Linear 4 Met Ala Ala Ala Ser Val Thr SerPro Gly Ser Leu Glu Leu Leu 1 5 10 15 Gln Pro Gly Phe Ser Lys Thr LeuLeu Gly Thr Arg Leu Glu Ala 20 25 30 Lys Tyr Leu Cys Ser Ala Cys Lys AsnIle Leu Arg Arg Pro Phe 35 40 45 Gln Ala Gln Cys Gly His Arg Tyr Cys SerPhe Cys Leu Thr Ser 50 55 60 Ile Leu Ser Ser Gly Pro Gln Asn Cys Ala AlaCys Val Tyr Glu 65 70 75 Gly Leu Tyr Glu Glu Gly Ile Ser Ile Leu Glu SerSer Ser Ala 80 85 90 Phe Pro Asp Asn Ala Ala Arg Arg Glu Val Glu Ser LeuPro Ala 95 100 105 Val Cys Pro Asn Asp Gly Cys Thr Trp Lys Gly Thr LeuLys Glu 110 115 120 Tyr Glu Ser Cys His Glu Gly Leu Cys Pro Phe Leu LeuThr Glu 125 130 135 Cys Pro Ala Cys Lys Gly Leu Val Arg Leu Ser Glu LysGlu His 140 145 150 His Thr Glu Gln Glu Cys Pro Lys Arg Ser Leu Ser CysGln His 155 160 165 Cys Arg Ala Pro Cys Ser His Val Asp Leu Glu Val HisTyr Glu 170 175 180 Val Cys Pro Lys Phe Pro Leu Thr Cys Asp Gly Cys GlyLys Lys 185 190 195 Lys Ile Pro Arg Glu Thr Phe Gln Asp His Val Arg AlaCys Ser 200 205 210 Lys Cys Arg Val Leu Cys Arg Phe His Thr Val Gly CysSer Glu 215 220 225 Met Val Glu Thr Glu Asn Leu Gln Asp His Glu Leu GlnArg Leu 230 235 240 Arg Glu His Leu Ala Leu Leu Leu Ser Ser Phe Leu GluAla Gln 245 250 255 Ala Ser Pro Gly Thr Leu Asn Gln Val Gly Pro Glu LeuLeu Gln 260 265 270 Arg Cys Gln Ile Leu Glu Gln Lys Ile Ala Thr Phe GluAsn Ile 275 280 285 Val Cys Val Leu Asn Arg Glu Val Glu Arg Val Ala ValThr Ala 290 295 300 Glu Ala Cys Ser Arg Gln His Arg Leu Asp Gln Asp LysIle Glu 305 310 315 Ala Leu Ser Asn Lys Val Gln Gln Leu Glu Arg Ser IleGly Leu 320 325 330 Lys Asp Leu Ala Met Ala Asp Leu Glu Gln Lys Val SerGlu Leu 335 340 345 Glu Val Ser Thr Tyr Asp Gly Val Phe Ile Trp Lys IleSer Asp 350 355 360 Phe Thr Arg Lys Arg Gln Glu Ala Val Ala Gly Arg ThrPro Ala 365 370 375 Ile Phe Ser Pro Ala Phe Tyr Thr Ser Arg Tyr Gly TyrLys Met 380 385 390 Cys Leu Arg Val Tyr Leu Asn Gly Asp Gly Thr Gly ArgGly Thr 395 400 405 His Leu Ser Leu Phe Phe Val Val Met Lys Gly Pro AsnAsp Ala 410 415 420 Leu Leu Gln Trp Pro Phe Asn Gln Lys Val Thr Leu MetLeu Leu 425 430 435 Asp His Asn Asn Arg Glu His Val Ile Asp Ala Phe ArgPro Asp 440 445 450 Val Thr Ser Ser Ser Phe Gln Arg Pro Val Ser Asp MetAsn Ile 455 460 465 Ala Ser Gly Cys Pro Leu Phe Cys Pro Val Ser Lys MetGlu Ala 470 475 480 Lys Asn Ser Tyr Val Arg Asp Asp Ala Ile Phe Ile LysAla Ile 485 490 495 Val Asp Leu Thr Gly Leu 500 501 44 amino acids AminoAcid Linear 5 Asp Leu Leu Cys Pro Ile Cys Met Gln Ile Ile Lys Asp AlaPhe 1 5 10 15 Leu Thr Ala Cys Gly His Ser Phe Cys Tyr Met Cys Ile IleThr 20 25 30 His Leu Arg Asn Lys Ser Asp Cys Pro Cys Cys Ser Gln His 3540 44 47 amino acids Amino Acid Linear 6 Glu Leu Ser Cys Ser Ile Cys LeuGlu Pro Phe Lys Glu Pro Val 1 5 10 15 Thr Thr Pro Cys Gly His Asn PheCys Gly Ser Cys Leu Asn Glu 20 25 30 Thr Trp Ala Val Gln Gly Ser Pro TyrLeu Cys Pro Gln Cys Arg 35 40 45 Ala Val 47 44 amino acids Amino AcidLinear 7 Leu Leu Arg Cys His Ile Cys Lys Asp Phe Leu Lys Val Pro Val 1 510 15 Leu Thr Pro Cys Gly His Thr Phe Cys Ser Leu Cys Ile Arg Thr 20 2530 His Leu Asn Asn Gln Pro Asn Cys Pro Leu Cys Leu Phe Glu 35 40 44 44amino acids Amino Acid Linear 8 Ala Phe Arg Cys His Val Cys Lys Asp PheTyr Asp Ser Pro Met 1 5 10 15 Leu Thr Ser Cys Asn His Thr Phe Cys SerLeu Cys Ile Arg Arg 20 25 30 Cys Leu Ser Val Asp Ser Lys Cys Pro Leu CysArg Ala Thr 35 40 44 45 amino acids Amino Acid Linear 9 Ser Ile Ser CysGln Ile Cys Glu His Ile Leu Ala Asp Pro Val 1 5 10 15 Glu Thr Asn CysLys His Val Phe Cys Arg Val Cys Ile Leu Arg 20 25 30 Cys Leu Lys Val MetGly Ser Tyr Cys Pro Ser Cys Arg Tyr Pro 35 40 45 45 amino acids AminoAcid Linear 10 Glu Val Thr Cys Pro Ile Cys Leu Asp Pro Phe Val Glu ProVal 1 5 10 15 Ser Ile Glu Cys Gly His Ser Phe Cys Gln Glu Cys Ile SerGln 20 25 30 Val Gly Lys Gly Gly Gly Ser Val Cys Ala Val Cys Arg Gln Arg35 40 45 46 amino acids Amino Acid Linear 11 Glu Leu Met Cys Pro Ile CysLeu Asp Met Leu Lys Asn Thr Met 1 5 10 15 Thr Thr Lys Glu Cys Leu HisArg Phe Cys Ser Asp Cys Ile Val 20 25 30 Thr Ala Leu Arg Ser Gly Asn LysGlu Cys Pro Thr Cys Arg Lys 35 40 45 Lys 46 50 amino acids Amino AcidLinear 12 Glu Val Thr Cys Pro Ile Cys Leu Glu Leu Leu Lys Glu Pro Val 15 10 15 Ser Ala Asp Cys Asn His Ser Phe Cys Arg Ala Cys Ile Thr Leu 2025 30 Asn Tyr Glu Ser Asn Arg Asn Thr Asp Gly Lys Gly Asn Cys Pro 35 4045 Val Cys Arg Val Pro 50 47 amino acids Amino Acid Linear 13 Glu ThrThr Cys Pro Val Cys Leu Gln Tyr Phe Ala Glu Pro Met 1 5 10 15 Met LeuAsp Cys Gly His Asn Ile Cys Cys Ala Cys Leu Ala Arg 20 25 30 Cys Trp GlyThr Ala Glu Thr Asn Val Ser Cys Pro Gln Cys Arg 35 40 45 Glu Thr 47 48amino acids Amino Acid Linear 14 Phe Gln Leu Cys Lys Ile Cys Ala Glu AsnAsp Lys Asp Val Lys 1 5 10 15 Ile Glu Pro Cys Gly His Leu Met Cys ThrSer Cys Leu Thr Ser 20 25 30 Trp Gln Glu Ser Glu Gly Gln Gly Ser Ser GlyCys Pro Phe Cys 35 40 45 Arg Cys Glu 48 28 amino acids Amino Acid Linear15 Gly Gly Phe Lys Leu Val Thr Cys Asp Phe Cys Lys Arg Asp Asp 1 5 10 15Ile Lys Lys Lys Glu Leu Glu Thr His Tyr Lys Thr Cys 20 25 28 26 aminoacids Amino Acid Linear 16 Gln Asp Leu Ala Val Cys Asp Val Cys Asn ArgLys Phe Arg His 1 5 10 15 Lys Asp Tyr Leu Arg Asp His Gln Lys Thr His 2025 26 28 amino acids Amino Acid Linear 17 Thr Gly Lys Tyr Pro Phe IleCys Ser Glu Cys Gly Lys Ser Phe 1 5 10 15 Met Asp Lys Arg Tyr Leu LysIle His Ser Asn Val His 20 25 28 28 amino acids Amino Acid Linear 18 ThrGly Glu Lys Pro Tyr Thr Cys Thr Val Cys Gly Lys Lys Phe 1 5 10 15 IleAsp Arg Ser Ser Val Val Lys His Ser Arg Thr His 20 25 28 28 amino acidsAmino Acid Linear 19 Arg Lys Lys Phe Pro His Ile Cys Gly Glu Cys Gly LysGly Phe 1 5 10 15 Arg His Pro Ser Ala Leu Lys Lys His Ile Arg Val His 2025 28 28 amino acids Amino Acid Linear 20 Ser Glu Glu Lys Pro Phe GluCys Glu Glu Cys Gly Lys Lys Phe 1 5 10 15 Arg Thr Ala Arg His Leu ValLys His Gln Arg Ile His 20 25 28 28 amino acids Amino Acid Linear 21 ProAsn Glu Gln Met Ala Gln Cys Pro Ile Cys Gln Gln Phe Tyr 1 5 10 15 ProLeu Lys Ala Leu Glu Lys Thr His Leu Asp Glu Cys 20 25 28 28 amino acidsAmino Acid Linear 22 Pro Asp Asp Gly Leu Val Ala Cys Pro Ile Cys Leu ThrArg Met 1 5 10 15 Lys Glu Gln Gln Val Asp Arg His Leu Asp Thr Ser Cys 2025 28 21 base pairs Nucleic Acid Single Linear 23 CCTTGTGCCT GCAGAGAGAAG 21 35 base pairs Nucleic Acid Single Linear 24 CTAGGTTAAC TTTCGGTGCTCCCCAGCAGG GTCTC 35 35 base pairs Nucleic Acid Single Linear 25CTAGGTTAAC TGGAGAAGGG GACCTGCTCG TCCTT 35 35 base pairs Nucleic AcidSingle Linear 26 CTAGGTTAAC TGCTGGCTTG GGAGGAGCAC TGTGA 35 35 base pairsNucleic Acid Single Linear 27 CTAGGTTAAC TGCTCCCGGT GCTGGCCCGG GCCTC 3534 base pairs Nucleic Acid Single Linear 28 CTAGGTTAAC TGCACTGGCCGAGCTCTCCA GGGA 34 15 base pairs Nucleic Acid Single Linear 29GTGATGAGAA TTCAT 15 21 base pairs Nucleic Acid Single Linear 30CGATGAATTC TCATCACTGC A 21 33 base pairs Nucleic Acid Single Linear 31GATCGGATCC AAAAAGAAGC CCTTGTGCCT GCA 33 16 base pairs Nucleic AcidSingle Linear 32 GCCTGGTTAA CTGGGC 16 19 base pairs Nucleic Acid SingleLinear 33 GCNCCNATGG CNYTNGARC 19 19 base pairs Nucleic Acid SingleLinear 34 GCNCCNATGG CNYTNGARA 19 19 base pairs Nucleic Acid SingleLinear 35 GCNCCNATGG CNYTNGARG 19 19 base pairs Nucleic Acid SingleLinear 36 GYTCNARNGC CATNGGNGC 19 19 base pairs Nucleic Acid SingleLinear 37 TYTCNARNGC CATNGGNGC 19 19 base pairs Nucleic Acid SingleLinear 38 CYTCNARNGC CATNGGNGC 19 17 base pairs Nucleic Acid SingleLinear 39 AARCAYGCNT AYGTNAA 17 17 base pairs Nucleic Acid Single Linear40 TTNACRTANG CRTGYTT 17 7 amino acids Amino Acid Linear 41 Ala Pro MetAla Leu Glu Arg 1 5 7 6 amino acids Amino Acid Linear 42 Lys His Ala TyrVal Lys 1 5 6 7 amino acids Amino Acid Linear 43 Pro Gly Ser Asn Leu GlySer 1 5 7 8 amino acids Amino Acid Linear 44 Lys Asp Asp Thr Met Phe LeuLys 1 5 8 37 base pairs Nucleic Acid Single Linear 45 TCGATCGTCGACCAAAAAGA AGCCCTCCTG CCTACAA 37 28 base pairs Nucleic Acid SingleLinear 46 CTAGAGATCT CAGGGGTCAG GCCACTTT 28 41 base pairs Nucleic AcidSingle Linear 47 CTAGAGATCT GTTAACTTTC GGTGCTCCCC AGCAGGGTCT C 41 41base pairs Nucleic Acid Single Linear 48 CTAGAGATCT GTTAACTGGAGAAGGGGACC TGCTCGTCCT T 41 41 base pairs Nucleic Acid Single Linear 49CTAGAGATCT GTTAACTGCT GGCTTGGGAG GAGCACTGTG A 41 34 base pairs NucleicAcid Single Linear 50 TCGATCGTCG ACCGCCTCCA GCTCAGCCCC TGAT 34 31 basepairs Nucleic Acid Single Linear 51 GATCGGATCC GGAGACACAG ATTCCAGCCC C31 35 base pairs Nucleic Acid Single Linear 52 GATCGAATTC TTAACTCTTCGGTGCTCCCC AGCAG 35 30 base pairs Nucleic Acid Single Linear 53GATCGGATCC TTGTGGTGTG TGGGGGTTGT 30 17 base pairs Nucleic Acid SingleLinear 54 CCTGGCTGGC CTAATGT 17 60 base pairs Nucleic Acid Single Linear55 GATCGACTCG AGATGCCCAA GAAGAAGCGG AAGGTGGCTG CAGCCAGTGT 50 GACTTCCCCT60 17 base pairs Nucleic Acid Single Linear 56 CTCTGGCGAA GAAGTCC 17 31base pairs Nucleic Acid Single Linear 57 GATCGGATCC GCCTCCAGCTCAGCCCCTGA T 31 31 base pairs Nucleic Acid Single Linear 58 GATCGGATCCAGCCAGCAGC TTCTCCTTCA C 31 30 base pairs Nucleic Acid Single Linear 59GATCGGATCC GCTCAGGCTC TTTTGGGGCA 30

1. An isolated tumor necrosis receptor associated factor (TRAF) capableof specific association with the intracellular domain of a native type 2TNF receptor (TNF-R2).
 2. The TRAF of claim 1 that is murine.
 3. TheTRAF of claim 1 that is capable of specific association with theintracellular domain of a native human TNF-R2.
 4. The TRAF of claim 1that is capable of specific binding to the intracellular domain of anative human TNF-R2.
 5. The TRAF of claim 1 that is native.
 6. The TRAFof claim 5 in homodimeric form.
 7. The TRAF of claim 5 associated withanother TRAF to form a heterodimer.
 8. The TRAF of claim 5 that is TRAF1(SEQ. ID. NO: 2) or TRAF2 (SEQ. ID. NO: 4).
 9. The TRAF of claim 1 whichcomprises a domain having at least about 50% sequence identity with theaa272-501 amino acid region of the TRAF2 amino acid sequence (SEQ. ID.NO: 4).
 10. The TRAF of claim 1 encoded by nucleic acid molecule capableof hybridizing, under stringent conditions, to the complement of thenucleotide sequence encoding amino acids 272-501 of the TRAF2 amino acidsequence (SEQ. ID. NO: 4).
 11. An isolated nucleic acid moleculecomprising a nucleotide sequence encoding a TRAF of claim
 1. 12. Avector comprising the nucleic acid molecule of claim 11 operably linkedto control sequences recognized by a host cell transformed with thevector.
 13. A host cell transformed with a vector of claim
 12. 14. Amolecule capable of disrupting the interaction of a TRAF and a nativeTNF-R2.
 15. An antibody capable of specific binding to a native TRAFpolypeptide.
 16. A hybridoma cell line producing an antibody of claim15.
 17. A method of using a nucleic acid molecule encoding a TRAFcomprising expressing such nucleic acid molecule in a cultured host celltransformed with a vector comprising such nucleic acid molecule operablylinked to control sequences recognized by said host cell, and recoveringthe polypeptide encoded by said nucleic acid molecule from the hostcell.
 18. A method for producing a TRAF polypeptide comprising insertinginto the DNA of a cell containing nucleic acid encoding said polypeptidea transcription modulatory element in sufficient proximity andorientation to the nucleic acid molecule to influence the transcriptionthereof.
 19. A method of determining the presence of a TRAF polypeptidecomprising hybridizing DNA encoding such polypeptide to a test samplenucleic acid and determining the presence of TRAF polypeptide DNA. 20.An isolated nucleic acid molecule encoding a fusion of an intracellulardomain sequence of a native TNF-R2 to the DNA-binding domain of atranscriptional activator.
 21. The nucleic acid molecule of claim 20,wherein said transcriptional activator is yeast GAL4.
 22. An isolatednucleic acid molecule encoding a fusion of a TRAF to the activationdomain of a transcriptional activator.
 23. The nucleic acid molecule ofclaim 22, wherein said transcriptional activator is yeast GAL4.
 24. Avector comprising the nucleic acid molecule of claim
 20. 25. A vectorcomprising the nucleic acid molecule of claim
 22. 26. An assay foridentifying a factor capable of specific binding to the intracellulardomain of a native TNF-R2, comprising (a) expressing nucleic acidmolecules encoding a polypeptide comprising a fusion of an intracellulardomain sequence of a native TNF-R2 to the DNA-binding domain of atranscriptional activator, and a fusion of a candidate polypeptidefactor to the activation domain of a transcriptional activator, in asingle host cell carrying a reporter gene; (b) monitoring the binding ofsaid candidate factor to the intracellular domain of TNF-R2 by detectinga signal of the molecule encoded by said reporter gene.
 27. As assay foridentifying a factor capable of specific association with theintracellular domain of a native TNF-R2, comprising (a) expressingnucleic acid molecules encoding a polypeptide comprising a fusion of anintracellular domain sequence of a native TNF-R2 to the DNA-bindingdomain of a transcriptional activator, and a fusion of a candidatefactor to the activation domain of a transcriptional activator, in asingle host cell transfected with nucleic acid encoding a polypeptidefactor capable of specific binding to said TNF-R2, and with nucleic acidencoding a reporter gene; and (b) monitoring the association of saidcandidate factor with said TNF-R2 or with said polypeptide factorcapable of specific binding to said TNF-R2 by detecting the signal ofthe polypeptide encoded by said reporter gene.
 28. A method ofamplifying a nucleic acid test sample, comprising priming a nucleic acidpolymerase reaction with nucleic acid encoding a TRAF polypeptidecapable of specific association with the intracellular domain of anative TNF-R2.
 29. A method for detecting a nucleic acid sequence codingfor a polypeptide molecule which comprises all or part of a TRAFpolypeptide or a related nucleic acid sequence, comprising contactingthe nucleic acid sequence with a detectable marker which bindsspecifically to at least part of said nucleic acid sequence, anddetecting the marker so bound.
 30. A method for the prevention ortreatment of a pathological condition associated with a TNF biologicalactivity mediated, fully or partially, by a TNF-R2, comprisingadministering to a patient in need a preventatively or therapeuticallyeffective amount of a TRAF or a molecule capable of disrupting theinteraction of a TRAF and said TNF-R2.
 31. An isolated human tumornecrosis factor receptor associated factor (TRAF) encoded by a DNAmolecule obtainable from a human recombinant cDNA library or genomic DNAlibrary (a) by the yeast two-hybrid technique or (b) by cross-specieshybridization with an oligonucleotide sequence derived from thenucleotide sequence encoding murine TRAF1 (SEQ. ID. NO: 1) or murineTRAF2 (SEQ. ID. NO: 3).
 32. An isolated human tumor necrosis factorreceptor associated factor (TRAF) which is the human homolog of themurine TNFR1 (SEQ. ID. NO: 2).
 33. An isolated human tumor necrosisfactor receptor associated factor (TRAF) which is the human homolog ofthe murine TNFR2 (SEQ. ID. NO: 4).